Magnetic resonance imaging-guided phase 1 trial of putaminal AADC gene therapy for Parkinson's disease.

OBJECTIVE: To understand the safety, putaminal coverage, and enzyme expression of adeno-associated viral vector serotype-2 encoding the complementary DNA for the enzyme, aromatic L-amino acid decarboxylase (VY-AADC01), delivered using novel intraoperative monitoring to optimize delivery. METHODS: Fifteen subjects (three cohorts of 5) with moderately advanced Parkinson's disease and medically refractory motor fluctuations received VY-AADC01 bilaterally coadministered with gadoteridol to the putamen using intraoperative magnetic resonance imaging (MRI) guidance to visualize the anatomic spread of the infusate and calculate coverage. Cohort 1 received 8.3 × 1011 vg/ml and ≤450 µl per putamen (total dose, ≤7.5 × 1011 vg); cohort 2 received the same concentration (8.3 × 1011 vg/ml) and ≤900 µl per putamen (total dose, ≤1.5 × 1012 vg); and cohort 3 received 2.6 × 1012 vg/ml and ≤900 µl per putamen (total dose, ≤4.7 × 1012 vg). (18)F-fluoro-L-dihydroxyphenylalanine positron emission tomography (PET) at baseline and 6 months postprocedure assessed enzyme activity; standard assessments measured clinical outcomes. RESULTS: MRI-guided administration of ascending VY-AADC01 doses resulted in putaminal coverage of 21% (cohort 1), 34% (cohort 2), and 42% (cohort 3). Cohorts 1, 2, and 3 showed corresponding increases in enzyme activity assessed by PET of 13%, 56%, and 79%, and reductions in antiparkinsonian medication of -15%, -33%, and -42%, respectively, at 6 months. At 12 months, there were dose-related improvements in clinical outcomes, including increases in patient-reported ON-time without troublesome dyskinesia (1.6, 3.3, and 1.5 hours, respectively) and quality of life. INTERPRETATION: Novel intraoperative monitoring of administration facilitated targeted delivery of VY-AADC01 in this phase 1 study, which was well tolerated. Increases in enzyme expression and clinical improvements were dose dependent. ClinicalTrials.gov Identifier: NCT01973543 Ann Neurol 2019;85:704-714.

Data-driven evolution of neurosurgical gene therapy delivery in Parkinson's disease.

Loss of nigrostriatal dopaminergic projection neurons is a key pathology in Parkinson's disease, leading to abnormal function of basal ganglia motor circuits and the accompanying characteristic motor features. A number of intraparenchymally delivered gene therapies designed to modify underlying disease and/or improve clinical symptoms have shown promise in preclinical studies and subsequently were evaluated in clinical trials. Here we review the challenges with surgical delivery of gene therapy vectors that limited therapeutic outcomes in these trials, particularly the lack of real-time monitoring of vector administration. These challenges have recently been addressed during the evolution of novel techniques for vector delivery that include the use of intraoperative MRI. The preclinical development of these techniques are described in relation to recent clinical translation in an adeno-associated virus serotype 2-mediated human aromatic L-amino acid decarboxylase gene therapy development programme. This new paradigm allows visualisation of the accuracy and adequacy of viral vector delivery within target structures, enabling intertrial modifications in surgical approaches, cannula design, vector volumes and dosing. The rapid, data-driven evolution of these procedures is unique and has led to improved vector delivery.

Aromatic L-Amino Acid Decarboxylase Gene Therapy Enhances Levodopa Response in Parkinson's Disease.

BACKGROUND: As Parkinson's disease progresses, levodopa treatment loses efficacy, partly through the loss of the endogenous dopamine-synthesizing enzyme L-amino acid decarboxylase (AADC). In the phase I PD-1101 study, putaminal administration of VY-AADC01, an investigational adeno-associated virus serotype-2 vector for delivery of the AADC gene in patients with advanced Parkinson's disease, was well tolerated, improved motor function, and reduced antiparkinsonian medication requirements. OBJECTIVES: This substudy aimed to determine whether the timing and magnitude of motor response to intravenous levodopa changed in PD-1101 patients after VY-AADC01 administration. METHODS: Participants received 2-hour threshold (0.6 mg/kg/h) and suprathreshold (1.2 mg/kg/h) levodopa infusions on each of 2 days, both before and approximately 6 months after VY-AADC01. Infusion order was randomized and double blinded. Unified Parkinson's Disease Rating Scale motor scores, finger-tapping speeds, and dyskinesia rating scores were assessed every 30 minutes for 1 hour before and ≥3 hours after start of levodopa infusion. RESULTS: Of 15 PD-1101 patients, 13 participated in the substudy. Unified Parkinson's Disease Rating Scale motor score area under the curve responses to threshold and suprathreshold levodopa infusions increased by 168% and 67%, respectively, after VY-AADC01; finger-tapping speeds improved by 162% and 113%, and dyskinesia scores increased by 208% and 72%, respectively, after VY-AADC01. Adverse events (mild/moderate severity) were reported in 5 participants during levodopa infusions pre-VY-AADC01 and 2 participants post-VY-AADC01 administration. CONCLUSIONS: VY-AADC01 improved motor responses to intravenous levodopa given under controlled conditions. These data and findings from the parent study support further clinical development of AADC gene therapy for people with Parkinson's disease. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Long-term RNAi knockdown of α-synuclein in the adult rat substantia nigra without neurodegeneration.

α-Synuclein plays a central role in the pathogenesis of Parkinson's disease (PD); interventions that decrease its expression appear neuroprotective in PD models. Successful translation of these observations into effective therapies will be dependent on the safety of suppressing α-synuclein expression in the adult brain. We investigated long-term α-synuclein knockdown in the adult rat CNS. 8-month old animals received either AAV-sh[Snca] (an RNA interference vector targeting the Snca mRNA transcript) or AAV-sh[Ctrl] (a control vector) unilaterally into the substantia nigra. No signs of systemic toxicity or motor dysfunction were observed in either experimental group over 12 months. Viral transgene expression persisted to 12 months post-inoculation, at which point Snca mRNA expression in substantia nigra dopaminergic neurons of animals that received AAV-sh[Snca] was decreased by ≈90%, and α-synuclein immunoreactivity by >70% relative to the control side. Stereological quantification of Nissl-labeled neurons showed no evidence of neurodegeneration in the substantia nigra 12 months after inoculation with either vector, and we observed abundant dopaminergic neurons with minimal α-synuclein immunoreactivity that appeared otherwise unremarkable in the AAV-sh[Snca] group. Despite the absence of neurodegeneration, some loss of TH expression was evident in nigral neurons after transduction with either vector, presumably a non-specific consequence of vector delivery, cellular transduction, or expression of shRNA or GFP. We conclude that long-term α-synuclein knockdown in the substantia nigra does not cause significant functional deficits in the ascending dopaminergic projection, or neurodegeneration. These findings are encouraging that it may be feasible to target α-synuclein expression therapeutically in PD.

Transient exposure to rotenone causes degeneration and progressive parkinsonian motor deficits, neuroinflammation, and synucleinopathy.

Individuals with Parkinson's disease (PD) typically receive a diagnosis once they have developed motor symptoms, at which point there is already significant loss of substantia nigra dopamine neurons, α-synuclein accumulation in surviving neurons, and neuroinflammation. Consequently, the point of clinical presentation may be too late to initiate disease-modifying therapy. In contrast to this clinical reality, animal models often involve acute neurodegeneration and potential therapies are tested concurrently or shortly after the pathogenic insult has begun rather than later when diagnostic clinical symptoms emerge. Therefore, we sought to develop a model that reflects the clinical situation more accurately. Middle-aged rats (7-9 months-old) received a single daily intraperitoneal injection of rotenone for 5 consecutive days and were observed over the next 8-9 months. Rotenone-treated rats showed transient motor slowing and postural instability during exposure but recovered within 9 days of rotenone cessation. Rats remained without behavioral deficits for 3-4 months, then developed progressive motor abnormalities over the ensuing months. As motor abnormalities began to emerge 3 months after rotenone exposure, there was significant loss of nigral dopaminergic neurons and significant microglial activation. There was delayed accumulation of α-synuclein in neurons of the substantia nigra and frontal cortex, which was maximal at 9 months post-rotenone. In summary, a brief temporally-remote exposure to rotenone causes delayed and progressive behavioral and neuropathological changes similar to Parkinson's disease. This model mimics the human clinical situation, in which pathogenesis is well-established by the time diagnostic motor deficits appear. As such, this model may provide a more relevant experimental system in which to test disease-modifying therapeutics.

Safety of AADC Gene Therapy for Moderately Advanced Parkinson Disease: Three-Year Outcomes From the PD-1101 Trial.

BACKGROUND AND OBJECTIVES: To report final, 36-month safety and clinical outcomes from the PD-1101 trial of NBIb-1817 (VY-AADC01) in participants with moderately advanced Parkinson disease (PD) and motor fluctuations. METHODS: PD-1101 was a phase 1b, open-label, dose escalation trial of VY-AADC01, an experimental AAV2 gene therapy encoding the human aromatic l-amino acid decarboxylase (AADC) enzyme. VY-AADC01 was delivered via bilateral, intraoperative MRI-guided putaminal infusions to 3 cohorts (n = 5 participants per cohort): cohort 1, ≤7.5 × 1011 vector genomes (vg); cohort 2, ≤1.5 × 1012 vg; cohort 3, ≤4.7 × 1012 vg. RESULTS: No serious adverse events (SAEs) attributed to VY-AADC01 were reported. All 4 non-vector-related SAEs (atrial fibrillation and pulmonary embolism in 1 participant and 2 events of small bowel obstruction in another participant) resolved. Requirements for PD medications were reduced by 21%-30% in the 2 highest dose cohorts at 36 months. Standard measures of motor function (PD diary, Unified Parkinson's Disease Rating Scale III "off"-medication and "on"-medication scores), global impressions of improvement (Clinical Global Impression of Improvement, Patient Global Impression of Improvement), and quality of life (39-item Parkinson's Disease Questionnaire) were stable or improved compared with baseline at 12, 24, and 36 months following VY-AADC01 administration across cohorts. DISCUSSIONS: VY-AADC01 and the surgical administration procedure were well-tolerated and resulted in stable or improved motor function and quality of life across cohorts, as well as reduced PD medication requirements in cohorts 2 and 3 over 3 years. TRIAL REGISTRATION INFORMATION: NCT01973543. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that, in patients with moderately advanced PD and motor fluctuations, putaminal infusion of VY-AADC01 is well tolerated and may improve motor function.

An Update on Gene Therapy Approaches for Parkinson's Disease: Restoration of Dopaminergic Function.

At present there is a significant unmet need for clinically available treatments for Parkinson's disease (PD) patients to stably restore balance to dopamine network function, leaving patients with inadequate management of symptoms as the disease progresses. Gene therapy is an attractive approach to impart a durable effect on neuronal function through introduction of genetic material to reestablish dopamine levels and/or functionally recover dopaminergic signaling by improving neuronal health. Ongoing clinical gene therapy trials in PD are focused on enzymatic enhancement of dopamine production and/or the restoration of the nigrostriatal pathway to improve dopaminergic network function. In this review, we discuss data from current gene therapy trials for PD and recent advances in study design and surgical approaches.

Gene Therapy in Movement Disorders: A Systematic Review of Ongoing and Completed Clinical Trials.

Introduction: We sought to provide an overview of the published and currently ongoing movement disorders clinical trials employing gene therapy, defined as a technology aiming to modulate the expression of one or more genes to achieve a therapeutic benefit. Methods: We systematically reviewed movement disorders gene therapy clinical trials from PubMed and ClinicalTrials.gov using a searching strategy that included Parkinson disease (PD), Huntington disease (HD), amino acid decarboxylase (AADC) deficiency, multiple system atrophy (MSA), progressive supranuclear palsy (PSP), dystonia, tremor, ataxia, and other movement disorders. Data extracted included study characteristics, investigational product, route of administration, safety/tolerability, motor endpoints, and secondary outcomes (i.e., neuroimaging, biomarkers). Results: We identified a total of 46 studies focusing on PD (21 published and nine ongoing), HD (2 published and 5 ongoing), AADC deficiency (4 published and 2 ongoing), MSA (2 ongoing), and PSP (1 ongoing). In PD, intraparenchymal infusion of viral vector-mediated gene therapies demonstrated to be safe and showed promising preliminary data in trials aiming at restoring the synthesis of dopamine, enhancing the production of neurotrophic factors, or modifying the functional interaction between different nodes of the basal ganglia. In HD, monthly intrathecal delivery of an antisense oligonucleotide (ASO) targeting the huntingtin protein (HTT) mRNA proved to be safe and tolerable, and demonstrated a dose-dependent reduction of the cerebrospinal fluid levels of mutated HTT, while a small phase-I study testing implantable capsules of cells engineered to synthesize ciliary neurotrophic factor failed to show consistent drug delivery. In AADC deficiency, gene replacement studies demonstrated to be relatively safe in restoring catecholamine and serotonin synthesis, with promising outcomes. Ongoing movement disorders clinical trials are focusing on a variety of gene therapy approaches including alternative viral vector serotypes, novel recombinant genes, novel delivery techniques, and ASOs for the treatment of HD, MSA, and distinct subtypes of PD (LRRK2 mutation or GBA1 mutation carriers). Conclusion: Initial phase-I and -II studies tested the safety and feasibility of gene therapy in PD, HD, and AADC deficiency. The ongoing generation of clinical trials aims to test the efficacy of these approaches and explore additional applications for gene therapy in movement disorders.

Gene therapy for Parkinson's disease: contemporary practice and emerging concepts.

INTRODUCTION: Gene therapy is a rapidly evolving technology that has predominantly utilized viral vectors to effectively deliver genetic material inside neurons to modulate the expression of one or more particular genes. Several gene therapy clinical trials have been conducted in Parkinson's disease (PD) by exploring strategies to either restore dopamine synthesis, enhance the production of trophic factors, enhance lysosomal function, or modify the interaction between different functional nodes of the basal ganglia. AREAS COVERED: In this review, the authors sought to discuss contemporary practice, emerging concepts, and unmet needs for the future of gene therapy in PD. EXPERT OPINION: While safety has been demonstrated, clinical trials on gene therapy for PD highlight the need for higher than anticipated volumes of infusion in order to optimize dose and vector coverage. Neurosurgical delivery techniques for gene therapy have rapidly evolved from the use of multiple trans-frontal trajectories to a single parietooccipital shape-conforming infusion. The employment of convection-enhanced delivery with reflux-resistant cannulas has further improved the vector diffusion into the target structures. Future technological developments will reduce the invasiveness and duration of surgery, improve specificity and transduction capacity with novel capsid designs, and implement strategies to control transgene expression.

GDNF and Parkinson's Disease: Where Next? A Summary from a Recent Workshop.

The concept of repairing the brain with growth factors has been pursued for many years in a variety of neurodegenerative diseases including primarily Parkinson's disease (PD) using glial cell line-derived neurotrophic factor (GDNF). This neurotrophic factor was discovered in 1993 and shown to have selective effects on promoting survival and regeneration of certain populations of neurons including the dopaminergic nigrostriatal pathway. These observations led to a series of clinical trials in PD patients including using infusions or gene delivery of GDNF or the related growth factor, neurturin (NRTN). Initial studies, some of which were open label, suggested that this approach could be of value in PD when the agent was injected into the putamen rather than the cerebral ventricles. In subsequent double-blind, placebo-controlled trials, the most recent reporting in 2019, treatment with GDNF did not achieve its primary end point. As a result, there has been uncertainty as to whether GDNF (and by extrapolation, related GDNF family neurotrophic factors) has merit in the future treatment of PD. To critically appraise the existing work and its future, a special workshop was held to discuss and debate this issue. This paper is a summary of that meeting with recommendations on whether there is a future for this therapeutic approach and also what any future PD trial involving GDNF and other GDNF family neurotrophic factors should consider in its design.

Unregulated cytosolic dopamine causes neurodegeneration associated with oxidative stress in mice.

The role of dopamine as a vulnerability factor and a toxic agent in Parkinson's disease (PD) is still controversial, yet the presumed dopamine toxicity is partly responsible for the "DOPA-sparing" clinical practice that avoids using L-3,4-dihydroxyphenylalanine (L-DOPA), a dopamine precursor, in early PD. There is a lack of studies on animal models that directly isolate dopamine as one determining factor in causing neurodegeneration. To address this, we have generated a novel transgenic mouse model in which striatal neurons are engineered to take up extracellular dopamine without acquiring regulatory mechanisms found in dopamine neurons. These mice developed motor dysfunctions and progressive neurodegeneration in the striatum within weeks. The neurodegeneration was accompanied by oxidative stress, evidenced by substantial oxidative protein modifications and decrease in glutathione. Ultrastructural morphologies of degenerative cells suggest necrotic neurodegeneration. Moreover, L-DOPA accelerated neurodegeneration and worsened motor dysfunction. In contrast, reducing dopamine input to striatum by lesioning the medial forebrain bundle attenuated motor dysfunction. These data suggest that pathology in genetically modified striatal neurons depends on their dopamine supply. These neurons were also supersensitive to neurotoxin. A very low dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (5 mg/kg) caused profound neurodegeneration of striatal neurons, but not midbrain dopamine neurons. Our results provide the first in vivo evidence that chronic exposure to unregulated cytosolic dopamine alone is sufficient to cause neurodegeneration. The present study has significant clinical implications, because dopamine replacement therapy is the mainstay of PD treatment. In addition, our model provides an efficient in vivo approach to test therapeutic agents for PD.

Outcomes of Interventional-MRI Versus Microelectrode Recording-Guided Subthalamic Deep Brain Stimulation.

In deep brain stimulation (DBS) of the subthalamic nucleus (STN) for Parkinson's disease (PD), there is debate concerning the use of neuroimaging alone to confirm correct anatomic placement of the DBS lead into the STN, versus the use of microelectrode recording (MER) to confirm functional placement. We performed a retrospective study of a contemporaneous cohort of 45 consecutive patients who underwent either interventional-MRI (iMRI) or MER-guided DBS lead implantation. We compared radial lead error, motor and sensory side effect, and clinical benefit programming thresholds, and pre- and post-operative unified PD rating scale scores, and levodopa equivalent dosages. MER-guided surgery was associated with greater radial error compared to the intended target. In general, side effect thresholds during initial programming were slightly lower in the MER group, but clinical benefit thresholds were similar. No significant difference in the reduction of clinical symptoms or medication dosage was observed. In summary, iMRI lead implantation occurred with greater anatomic accuracy, in locations demonstrated to be the appropriate functional region of the STN, based on the observation of similar programming side effect and benefit thresholds obtained with MER. The production of equivalent clinical outcomes suggests that surgeon and patient preference can be used to guide the decision of whether to recommend iMRI or MER-guided DBS lead implantation to appropriate patients with PD.

LRRK2 activation in idiopathic Parkinson's disease.

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD). However, a potential role of wild-type LRRK2 in idiopathic PD (iPD) remains unclear. Here, we developed proximity ligation assays to assess Ser1292 phosphorylation of LRRK2 and, separately, the dissociation of 14-3-3 proteins from LRRK2. Using these proximity ligation assays, we show that wild-type LRRK2 kinase activity was selectively enhanced in substantia nigra dopamine neurons in postmortem brain tissue from patients with iPD and in two different rat models of the disease. We show that this occurred through an oxidative mechanism, resulting in phosphorylation of the LRRK2 substrate Rab10 and other downstream consequences including abnormalities in mitochondrial protein import and lysosomal function. Our study suggests that, independent of mutations, wild-type LRRK2 plays a role in iPD. LRRK2 kinase inhibitors may therefore be useful for treating patients with iPD who do not carry LRRK2 mutations.

Alpha-synuclein inhibits aromatic amino acid decarboxylase activity in dopaminergic cells.

Alpha-synuclein is a presynaptic protein strongly implicated in Parkinson's disease (PD). Because dopamine neurons are invariably compromised during pathogenesis in PD, we have been exploring the functions of alpha-synuclein with particular relevance to dopaminergic neuronal cells. We previously discovered reduced tyrosine hydroxylase (TH) activity and minimal dopamine synthesis in stably-transfected MN9D cells overexpressing either wild-type or A53T mutant (alanine to threonine at amino acid 53) alpha-synuclein. TH, the rate-limiting enzyme in dopamine synthesis, converts tyrosine to l-dihydroxyphenylalanine (L-DOPA), which is then converted to dopamine by the enzyme, aromatic amino acid decarboxylase (AADC). We confirmed an interaction between alpha-synuclein and AADC in striatum. We then sought to determine whether wild-type or A53T mutant alpha-synuclein might have affected AADC activity in dopaminergic cells. Using HPLC with electrochemical detection, we measured dopamine and related catechols after L-DOPA treatments to bypass the TH step. We discovered that while alpha-synuclein did not reduce AADC protein levels, it significantly reduced AADC activity and phosphorylation in our cells. These novel findings further support a role for alpha-synuclein in dopamine homeostasis and may explain, at least in part, the selective vulnerability of dopamine neurons that occurs in PD.

Erythropoietin protects against 6-hydroxydopamine-induced dopaminergic cell death.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death of midbrain dopaminergic neurons. In the present study, erythropoietin, a trophic factor that has both hematopoietic and neural protective characteristics, was investigated for its capacity to protect dopaminergic neurons in experimental Parkinson's disease. Using both the dopaminergic cell line, MN9D, and primary dopamine neurons, we show that erythropoietin (1-3 U/mL) is neuroprotective against the dopaminergic neurotoxin, 6-hydroxydopamine. Protection was mediated by the erythropoietin receptor, as neutralizing anti-erythropoietin receptor antibody abrogated the protection. Activation of Akt/protein kinase B (PKB), via the phosphoinositide 3-kinase pathway, is a critical mechanism in erythropoietin-induced protection, while activation of extracellular signal-regulated kinase (ERK)1/2 contributes only moderately. Indeed, transfection of constitutively active Akt/PKB into dopaminergic cells was sufficient to protect against cell death. Furthermore, erythropoietin diminished markers of apoptosis in MN9D cells, including caspase 9 and caspase 3 activation and internucleosomal DNA fragmentation, suggesting that erythropoietin interferes with the apoptosis-execution process. When erythropoietin was administered to mice unilaterally lesioned with 6-hydroxydopamine, it prevented the loss of nigral dopaminergic neurons and maintained striatal catecholamine levels for at least 8 weeks. Erythropoietin-treated mice also had significantly reduced behavioral asymmetries. These studies suggest that erythropoietin can be an effective neuroprotective agent for dopaminergic neurons, and may be useful in reversing behavioral deficits associated with Parkinson's disease.