Gene Therapy in Parkinson’s Disease: Rationale and Current Status

1. Parkinson’s Disease

The second most common age-related progressive neurodegenerative disorder, PD, first described in 1817 by James Parkinson in his “An Essay on the Shaking Palsy”, affects 1.5 million people in the United States and 4 million people worldwide.(1, 2) Approximately 1–2% of the population over the age of 65 years suffers from PD and this number is expected to grow as the average age of the population increases.(3) The cardinal clinical features include: tremor, cogwheel rigidity, akinesia, bradykinesia and postural instability. However, there is a spectrum among PD patients in terms of clinical manifestations and disease progression. For example, while most patients exhibit the “typical” PD tremor, a rhythmic back-and-forth movement in the range of 4–6 Hz commonly present in the hands or limbs when the patient is at rest (i.e. resting tremor; hands supported against gravity), the severity of these motoric symptoms varies with a subset of PD patients classified as having a tremor-predominant PD phenotype. Although most recognized by the aforementioned debilitating motor dysfunctions, PD patients can also suffer from a variety of non-motor symptoms, including; depression, cognitive impairment, psychosis, hallucinations, compulsive behaviors, REM sleep behavior disorders, excessive daytime somnolence, orthostatic hypotension, gastrointestinal symptoms, constipation, urinary and sexual dysfunction, speech changes, skin problems, pain, difficulty swallowing and chewing.(4–8) Several of these non-motor complications such as those associated with cognition arise later in disease (Hoehn and Yahr Stages IV–V) while a subset (i.e. gastrointestinal system dysfunction) are prodromal, preceding the motor manifestations by as much as 10 years.(8–10)

PD is characterized by the loss of substantia nigra pars compacta (SNpc) dopamine neurons, dystrophy of the associated projection fibers to the corpus striatum and diminution of the nigrostriatal neurotransmitter, dopamine. In fact, motoric symptoms are underappreciated until approximately 60% of the SNpc dopamine neurons are lost resulting in a greater than 80% loss of dopamine.(11, 12) The pathological hallmark of PD is presence of intracytoplasmic proteinaceous inclusions called Lewy bodies, which are localized most notably to the few surviving SNpc dopaminergic neurons and were first described by Friederich Lewy in 1912. The function of Lewy bodies in PD pathobiology is not understood. Nonetheless, following immunohistochemical staining with antibodies against two abundant Lewy body proteins, ubiquitin and α-synuclein, the presence of these structures in the remaining SNpc neurons is used as a criteria for diagnosis of PD.(13, 14) The spatial and temporal localization of Lewy bodies has recently been established as a marker for different neuropathological stages of PD. Data from this study demonstrate that Lewy body pathology in PD subjects does not begin in nor is it restricted to the SNpc which corresponds with the observation that non-motor symptoms are also present in this disease.(15) Another feature revealed in PD brains upon autopsy is the presence of activated microglia.(16–18) As the immune surveillance cells of the brain, it is not surprising that microglia are upregulated in many neurodegenerative disorders following substantial cell death. However, evidence from both tissue culture and animal models of PD suggest that microglial activation and the consequent increase in proinflammatory molecules may play an important role in the progression of PD, representing another potential therapeutic avenue.(19–23)

It has been nearly 200 years since PD was first described and yet the initiating mechanism for sporadic disease has not been fully elucidated. Risk factors including advancing age, exposure to environmental toxicants and drinking well water have all been linked to PD, yet the vast majority of cases are sporadic with no known etiology.(3, 24–31) Genetic links to PD have been identified and form the basis for several animal models of this disease.(32–35) The first discovered PD-associated gene, SNCA, is localized to chromosome 4 and encodes a 16kDa protein called α-synuclein.(33, 36) Mutations within this gene were linked with an early onset familial form of PD.(34, 37) The importance of α-synuclein in both familial and sporadic PD etiology was further demonstrated following the discovery that duplication or triplication of SNCA was sufficient to cause PD suggesting that overexpression of α-synuclein alone leads to toxicity.(38, 39) Other PD-related genes that have been identified include: parkin, ubiquitin carboxyl-terminal esterase L1 (UCHL1), Leucine-Rich Repeat Kinase 2 (LRRK2), PTEN (phosphatase and tensin homolog)-induced kinase 1 (PINK1) and DJ-1.(35, 40–45) Of these genes, LRRK2 mutations are the most frequent and have been linked to both late-onset familial and sporadic PD.(46–48) Recent genome-wide association studies revealed several signals associated with PD, one in the gene coding α-synuclein, demonstrating a role for genetic variants in sporadic PD.(49, 50) However, even when taken together, genetic mutations alone do not account for the majority of sporadic PD cases and it has now been generally accepted that a combination of genetic vulnerability and environmental insults lead to sporadic PD.(51) Current clinical diagnosis of PD does not distinguish between initiating factors and usually occurs only after the invariant loss of SNpc dopamine neurons has reached a disease threshold or tipping point. Consequently, existing pharmacotherapies have been focused on enhancing the dysfunctional dopaminergic system, a feature common to all PD patients, rather than halting the disparate initiating events.

2. Rationale for Gene Therapeutic Approaches

All commonly employed PD therapies are focused on the amelioration of symptoms and do not cure disease.(52–71) Most pharmaceutical approaches focus on augmentation of the diminished neurotransmitter, dopamine, by either increasing production, extending half-life and/or decreasing metabolism. Dopamine is normally synthesized from the amino acid tyrosine in a series of enzymatic reactions initiated by the conversion of tyrosine to L-dopa by tyrosine hydroxylase and its co-factor tetrahydrobiopterin. L-dopa is then decarboxylated by L-amino acid decarboxylase (AADC) to form dopamine. Dopamine enhancement therapies are most effective when a portion of the nigrostriatal pathway is intact. Consequently, as the number of SNpc dopamine neurons and projection fibers decreases these treatments become less efficacious. The most common therapy for PD is the oral administration of the dopamine precursor, levodopa (L-dopa), usually taken in conjunction with an inhibitor of extracerebral dopa decarboxylase to prevent peripheral metabolism. Introduced more than 40 years ago, this treatment effectively quells the motoric symptoms of akinesia and bradykinesia.(52) However, many patients gradually develop levodopa-induced dyskinesias and motor fluctuations about 5–15 years after the initiation of L-dopa treatment.(72, 73) The severity of the dyskinesias varies between patients and ongoing research efforts are focused on the development of new and more effective anti-dyskinetic medications.(74) A subset of these patients benefit from deep brain stimulation (DBS) which is used in conjunction with L-dopa treatment. Although the exact mechanism of action for DBS is not clear, it is proposed that the generated impulses suppress neural activity, which in the case of subthalamic nucleus DBS (STN-DBS), dampens the PD-increased STN abnormal activity resulting in an overall improvement of tremors, rigidity and akinesias.(54, 57, 60, 62, 65, 69–71, 75–79) Often DBS-treated patients are able to reduce their L-dopa dose by ~50–60% resulting in a decrease in dyskinesias and report an improved quality of life.(71, 76, 77, 79) Unfortunately, much like L-dopa therapy, the efficacy of DBS declines as PD progresses.

There is no debate regarding the need for novel disease modifying PD therapies. New PD treatments are currently in clinical trials and several are centered on gene therapeutic approaches to either compensate for the loss of dopamine or to protect SNpc dopamine neurons from further degeneration with the overall goal of restoring function (see <clinicaltrials.gov>; Table I; reviewed in (80, 81)). There are several reasons for pursuing a viral vector-mediated gene therapeutic approach in the context of PD; 1) since the PD pathophysiology that subserves the motoric symptoms is largely confined to one brain region, the nigrostriatal pathway, a limited area will require treatment; 2) because of the physically restricted environment of the brain, repeated injections into the nigrostriatum are not desirable, making long-term gene expression following a single treatment appealing; 3) viral vectors are diffusible and theoretically capable of efficient transduction of the striatum; 4) genes have been identified that can either modulate the neuronal phenotype or act as neuroprotective agents; and 5) there is currently no cure for this debilitating disease.

3. Parkinson’s Disease Gene Therapy Platforms

The earliest attempts at PD gene therapy utilized a variety of cell and tissue transplants including fetal and autologous adrenal medullary tissue grafts, xenografts, neurospheres, cell suspension grafts and embryonic stem cells with the overall goal of augmenting dopamine content.(82–88) Fetal nerve cell transplantation has been met with some success as a subset of treated patients experienced palliative relief for many years. The effects of long-term fetal implants have recently been evaluated and upon autopsy several subjects displayed PD pathology in the grafted tissue suggesting that the local “disease” environment within the brain brings about “de novo” PD.(89–92) However, since there have been only a small number of treated patients available for evaluation it is difficult to draw any definitive conclusions regarding the apparent “transfer of disease”. Regardless these patients demonstrate the potential of cell replacement as an option to stave off disease for several years. Currently one of the biggest obstacles for fetal implant therapy is the acquisition of tissue.

The advent of viral vector technology revolutionized the gene therapy field providing a method for the efficient delivery of genetic material (transgene) without the need for human transplant tissue or cells. Although many different viral platforms have been developed, based on the criterion of safety, stability of gene expression and transduction of specific cellular targets only adeno-associated virus (AAV) and lentivirus vectors are currently in clinical trials for PD (see Table I; for review see (93)). These vectors share the common features of efficient transduction for both dividing and non-dividing cells and long-term transgene expression making them valuable for post-mitotic neuron-targeted therapies.

4. Parkinson’s Disease Gene Therapy Clinical Trials

Current PD gene therapy clinical trials employ either rAAV2 or lentivirus and are focused on three therapeutic approaches; augmentation of dopamine levels via increased neurotransmitter production, modulation of the neuronal phenotype, and neuroprotection. (106, 108–113) One approach focuses on increasing dopamine production via direct delivery of genes involved in neurotransmitter synthesis while a second method is designed to change the neuronal phenotype bypassing the need for dopamine, both approaches should the ameliorate symptoms associated with PD. These therapies are also intended to delay development of end-stage disease, an important accomplishment in a progressive age-related disorder such as PD. On the other hand, delivery of a neurotrophin gene such as glial cell-derived neurotrophic factor (GDNF) or neurturin (NTN), a GDNF-related protein, is projected to slow disease progression by enhancing neuronal survival. However in the long-term it is not known whether neurotrophins, while demonstrated to protect neurons in animal toxicant models of SNpc dopamine neuronal death, will represent a cure for PD. One weakness, which applies to all current PD therapeutic approaches is the lack of a reliable clinical test affording early diagnosis of PD patients prior to the development of motoric symptoms and substantial SNpc dopamine neuron loss.

5. Conclusion

Recent therapeutic advances have been reported for another neurodegenerative disease, adrenoleukodystrophy (ALD), using hematopoietic stem cells transduced with a lentiviral vector expressing the adenosine triphosphate-binding cassette transporter (ABCD1 gene).(145) These researchers report that ALD progression was halted following treatment. This work supports the concept that neurodegenerative diseases are amenable to gene therapeutic approaches. Although only incremental clinical advancements have been realized thus far for PD gene therapy there is hope that this technology will soon be an important addition to current therapeutic approaches. Gene therapy affords PD clinicians the opportunity to permanently alter dopamine production and neuronal phenotype. Even in the absence of a cure these types of therapies would represent significant therapeutic advancements.

There are critical issues which are currently being addressed that should facilitate the success of PD gene therapy.(133, 142) For example, viral vectors are consistently undergoing improved engineering to enhance delivery, diffusion, and regulated spatial and temporal transgene expression.(104, 146, 147) In addition, effective novel non-invasive imaging techniques to monitor transgene expression and diffusion of viral vectors are under development and will greatly aid the gene therapy field. (148, 149) Furthermore, age-related neurodegenerative diseases such as PD are in need of specific easily accessible and accurate biomarkers that will allow for early diagnosis as well as monitoring of disease progression; these types of studies are currently underway.(112, 133, 150)

It is not clear why preclinical gene therapy studies are more successful than the ensuing clinical trials. Some of the discrepancies could arise from the many different animal models that used for preclinical studies. Researchers use both neurotoxicant and genetic models that do not faithfully recapitulate the progressive degeneration and behavioral aspects of PD. Current work in the field is focused on establishing improved robust animal models. Another important and sometimes overlooked concept is that sporadic PD may represent a syndrome of diseases and therefore therapies may need to be tailored to specific parkinsonian subtypes.(41, 151) Both familial and sporadic PD arise from disparate initiating factors yet the final pathological hallmark is loss of SNpc dopamine neurons. Currently the path between initiation of disease and SNpc dopamine neuron death represents a continuum but with the identification of novel biomarkers clinicians may be able to separate PD patients into subtypes that will benefit from personalized therapies. Furthermore, consistent clinical trial criteria and outcome measurements would allow for reliable analysis of trial data. For example, different PD clinical trials using the same therapeutic molecule (i.e. GDNF) are difficult to interpret because of a lack of standard protocols.(133) Therefore establishing a uniform study design including the same viral vector serotype, injection paradigm, patient selection criteria and outcome measurements would allow for more consistent data interpretation. Finally, PD gene therapy may also benefit from multifaceted approaches, for example gene therapy cocktails such as rAAV2-NTN combined with rAAV2-hAADC and peripheral levodopa may improve both neuronal survival and function better than each treatment alone. In conclusion, as novel targets are identified and the hurdles mentioned above overcome, we anticipate that viral vector-mediated gene therapeutic approaches will have a profound impact on PD progression and outcome.