Human Aromatic L-amino Acid Decarboxylase Research Articles

AAV2-hAADC (Eladocagene Exuparvovec) Biodistribution and Expression: Superiority of Intraputaminal versus Intracerebroventricular and Intrathecal (Lumbar) Routes of Administration.

Aromatic L-amino acid decarboxylase deficiency is a genetic disorder of enzyme loss with decreased neurotransmitter synthesis, and it is characterized by symptoms of impaired motor development and cognitive function, hypotonia, dystonia, and oculogyric crises. Though symptomatic severity varies, the majority of patients experience severe motor impairments, including an inability to sit, stand, or walk. One approved therapy for Aromatic L-amino acid decarboxylase deficiency involves intraputaminal delivery of an adeno-associated virus packaging the human Aromatic L-amino acid decarboxylase enzyme (hAADC) cDNA. The objective of this study in monkeys was to determine the acceptability of ICV/IT as minimally invasive dosing options by evaluating hAADC biodistribution and expression following intraputaminal, intracerebroventricular (ICV), or intrathecal (IT, lumbar) administration. Results show that all routes produced comparable CSF transgene levels and were well-tolerated. The intraputaminal route yielded the highest levels of transgene-derived mRNA expression in the putamen, caudate, and globus pallidus, while expression levels in the spinal cord and dorsal root ganglia (DRG, a target of special toxicological concern) were undetectable. In contrast, the highest transgene levels in ICV/IT groups were observed in the spinal cord and DRG, but levels were too low to result in expression in the putamen, caudate, and globus pallidus. Unlike ICV/IT, the intraputaminal route produced no transgene in blood, suggesting a lower likelihood of off-target toxicities. Additionally, intraputaminal dosing resulted in the lowest anti-AAV2 antibody (anti-drug antibody) levels. Together, these data demonstrate the superiority of intraputaminal administration over ICV/IT routes in achieving AAV2-hAADC transgene DNA distribution and mRNA expression in target therapeutic areas while minimizing risk of toxicity.

Eladocagene Exuparvovec: First Approval.

Eladocagene exuparvovec (Upstaza™) is a gene therapy developed by PTC Therapeutics for the treatment of human aromatic L-amino acid decarboxylase (AADC) deficiency. Eladocagene exuparvovec comprises an adeno-associated virus vector that delivers the dopa decarboxylase (DDC) gene, the gene for human AADC. Eladocagene exuparvovec was approved in July 2022 in the EU for the treatment of patients aged 18 months and older with a clinical, molecular, and genetically confirmed diagnosis of AADC deficiency with a severe phenotype (i.e. patients who cannot sit, stand or walk). This article summarizes the milestones in the development of eladocagene exuparvovec leading to this first approval for the treatment of patients aged 18 months and older with AADC deficiency.

α-synuclein enfolds tyrosine hydroxylase and dopamine ß-hydroxylase, potentially reducing dopamine and norepinephrine synthesis.

Parkinson's disease (PD) results from degeneration of dopamine and norepinephrine neurons due to α-synuclein aggregates that likely have their origin in the gut. Tyrosine hydroxylase (TH) catalyses the formation of L-DOPA, the rate-limiting step in the biosynthesis of dopamine. A second enzyme, DOPA decarboxylase (DDC), catalyzes the conversion of L-DOPA to dopamine. A third enzyme, dopamine ß-hydroxylase (DBH), catalyzes the conversion of dopamine to norepinephrine. To analyze possible interactions of α-synuclein with TH, DDC and DBH, we performed in silico protein-protein docking. Protein data bank (pdb) entries were searched on the RCSB Protein Data Bank. We identified four structures that allowed us to examine the relationship of α-synuclein with TH, DDC, and DBH: (1) Human micelle-bound alpha-synuclein, (2) solution structure of the regulatory domain of tyrosine hydroxylase (Rattus norvegicus), (3) crystal structure of human aromatic L-amino acid decarboxylase (DOPA decarboxylase) in the apo form and (4) crystal structure of human dopamine ß-hydroxylase at 2.9 angstrom resolution. We used the ClusPro server (https://cluspro.org) for protein-protein docking. The protein structures were visualized with PyMOL v 2.3.4. α-synuclein partially enfolds tyrosine hydroxylase and dopamine ß-hydroxylase, potentially reducing dopamine and norepinephrine synthesis. α-synuclein may dock too far away from DOPA decarboxylase to affect its function directly. Our in silico finding of α-synuclein partly enfolding tyrosine hydroxylase and dopamine ß-hydroxylase suggests that α-synuclein docking inhibition could increase dopamine and norepinephrine biosynthesis, ameliorating PD symptoms. Small molecules that bind to α-synuclein have already been identified. Further studies may lead to new small molecule drugs that block α-synuclein enfolding of tyrosine hydroxylase and dopamine ß-hydroxylase.

Long-term efficacy and safety of eladocagene exuparvovec in patients with AADC deficiency

Aromatic L-amino acid decarboxylase deficiency results in decreased neurotransmitter levels and severe motor dysfunction. Twenty-six patients without head control received bilateral intraputaminal infusions of a recombinant adeno-associated virus type 2 vector containing the human aromatic L-amino acid decarboxylase gene (eladocagene exuparvovec) and have completed 1-year evaluations. Rapid improvements in motor and cognitive function occurred within 12months after gene therapy and were sustained during follow-up for>5 years. An increase in dopamine production was demonstrated by positron emission tomography and neurotransmitter analysis. Patient symptoms (mood, sweating, temperature, and oculogyric crises), patient growth, and patient caretaker quality of life improved. Although improvements were observed in all treated participants, younger age was associated with greater improvement. There were no treatment-associated brain injuries, and most adverse events were related to underlying disease. Post-surgery complications such as cerebrospinal fluid leakage were managed with standard of care. Most patients experienced mild to moderate dyskinesia that resolved in a few months. These observations suggest that eladocagene exuparvovec treatment for aromatic L-amino acid decarboxylase deficiency provides durable and meaningful benefits with a favorable safety profile.

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

Background and ObjectivesTo 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.MethodsPD-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.ResultsNo 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.DiscussionsVY-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 InformationNCT01973543.Classification of EvidenceThis 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.

Efficacy and safety of AAV2 gene therapy in children with aromatic L-amino acid decarboxylase deficiency: an open-label, phase 1/2 trial

Aromatic l-amino acid decarboxylase (AADC) deficiency is an inherited disease that causes depletion of neurotransmitters and severe motor dysfunction in infants and children. We previously reported compassionate use of an adeno-associated virus (AAV) vector containing the human AADC gene (AAV2-hAADC) in four children with AADC deficiency (aged 4-6 years). In this study, we aimed to establish the efficacy and safety of this treatment. We did an open-label, phase 1/2 trial at the National Taiwan University Hospital (Taipei, Taiwan). We included patients who had a definitive diagnosis and clinical symptoms of AADC deficiency (hypotonia, dystonia, and oculogyric crisis), who were older than 24 months or had skull bones suitable for stereotactic surgery, and who had an anti-AAV2 antibody titre lower than 1·0 optical density. All patients received bilateral intraputaminal injections of AAV2-hAADC (1·81 × 1011 vg in total) through stereotactic brain surgery. Primary efficacy outcomes were an increase in the Peabody Developmental Motor Scales (second edition; PDMS-2) score of greater than 10 points and an increase in homovanillic acid (HVA) or 5-hydroxyindoleacetic acid (5-HIAA) concentrations in the cerebrospinal fluid 12 months after gene therapy. We assessed patients at baseline and at 3, 6, 9, and 12 months after gene therapy, and every 6 months thereafter for one further year; all patients who received the treatment were included in the analysis. We assessed for surgical complications (cerebrospinal fluid leakage and intracerebral haemorrhage) at days 3-7 after AAV2 gene therapy, and we assessed adverse events during the follow-up evaluations for 12 months. This study is registered with ClinicalTrials.gov, number NCT01395641. Ten patients (median age 2·71 years, IQR 2·46-6·35) were enrolled from Oct 1, 2014, to Dec 2, 2015. All patients tolerated the surgeries and vector injections. One patient died from influenza B encephalitis during an endemic outbreak 10 months after treatment; therefore, 9 months of data were included in the analyses for this patient. All patients met the primary efficacy endpoint: 12 months after gene therapy, PDMS-2 scores were increased by a median of 62 points (IQR 39-93; p=0·005) and HVA concentrations by a median of 25 nmol/L (IQR 11-48; p=0·012); however, there was no significant change in 5-HIAA concentrations (median difference 0, IQR 0-5; p=0·20). In total, 101 adverse events were reported, with the most common being pyrexia (16 [16%] of 101 events) and orofacial dyskinesia (ten [10%]). 12 serious adverse events occurred in six patients, including one death (treatment-unrelated encephalitis due to influenza B infection), one life-threatening pyrexia, and ten events that led to hospital admission. Transient post-gene therapy dyskinesia occurred in all patients but was resolved with risperidone. Of 31 treatment-related adverse events, only one (patient 1) was severe in intensity, and none led to hospital admission or death. Our findings suggest that intraputaminal injection of AAV2-hAADC is well tolerated and might improve motor development in children with AADC deficiency. AADC Research Fund at National Taiwan University Hospital and the National Research Programme for Biopharmaceuticals.

A pathogenic S250F missense mutation results in a mouse model of mild aromatic l-amino acid decarboxylase (AADC) deficiency.

Homozygous mutations in the aromatic l-amino acid decarboxylase (AADC) gene result in a severe depletion of its namesake protein, triggering a debilitating and often fatal form of infantile Parkinsonism known as AADC deficiency. AADC deficient patients fail to produce normal levels of the monoamine neurotransmitters dopamine and serotonin, and suffer a multi-systemic disorder characterized by movement abnormalities, developmental delay and autonomic dysfunction; an absolute loss of dopamine is generally considered incompatible with life. There is no optimal treatment for AADC deficiency and few truly good models in which to investigate disease mechanisms or develop and refine therapeutic strategies. In this study, we introduced a relatively frequently reported but mildly pathogenic S250F missense mutation into the murine Aadc gene. We show that mutants homozygous for the mutation are viable and express a stable but minimally active form of the AADC protein. Although the low enzymatic activity of the protein resulted in only modestly reduced concentrations of brain dopamine, serotonin levels were markedly diminished, and this perturbed behavior as well as autonomic function in mutant mice. Still, we found no evidence of morphologic abnormalities of the dopaminergic cells in mutant brains. The striatum as well as substantia nigra appeared normal and no loss of dopamine expressing cells in the latter was detected. We conclude that even minute levels of active AADC are sufficient to allow for substantial amounts of dopamine to be produced in model mice harboring the S250F mutation. Such mutants represent a novel, mild model of human AADC deficiency.

C-11. An Update on Gene Therapy for the Treatment of Aromatic L-Amino Acid Decarboxylase (AADC) Deficiency

Aromatic L-amino acid decarboxylase (AADC) is an enzyme responsible for the final step of the synthesis of neurotransmitters dopamine and serotonin. AADC deficiency is a rare genetic disorder causing motor and autonomic dysfunction and early death in children. Taiwanese carry a high prevalence of AADC deficiency due to a founder mutation (IVS6+4A>T) in the AADC gene. A gene therapy with recombinant adeno-associated virus (AAV) serotype 2 CMV promoter-driven human AADC (AAV2-hAADC) was conducted in children with AADC deficiency since 2010. Clinical grade vector was manufactured. Two target points were determined in each putamen and a total of 1.7×1011 vg vector was injected. Eight patients have been treated at a mean age of 4.9 years (2-8 years), and the mean follow up period was 3.8 years (3-4.5 years). All patients, except one, had homozygous IVS6+4A>T mutations. Before gene therapy, these patients didn't have head control, and could not sit or stand, and they all had regular and severe oculogyric crisis (OGC) attacks. The stereotaxic surgeries were performed smoothly and there was no intracerebral bleeding. At the most recently follow-up visits after gene therapy, one patient was learning walking, two stood with support, one sat without assist, and three had better head control. The severity of OGC decreased and emotion stabilized in all patients.[18F]fluoro-dopa positron emission tomography (PET) signal intensity over putamen increased and cerebrospinal fluid neurotransmitter levels rose in all treated patients. Post gene therapy transient (orofacial) dyskinesia was the main adverse event of the treatment, and one patient experienced a hypovolemic shock due to dyskinesia and he remained bed-ridden. There is no virus-associated side effect, and currently no patient reveals a loss of treatment effect.

Carbidopa-based modulation of the functional effect of the AAV2-hAADC gene therapy in 6-OHDA lesioned rats.

Progressively blunted response to L-DOPA in Parkinson’s disease (PD) is a critical factor that complicates long-term pharmacotherapy in view of the central importance of this drug in management of the PD-related motor disturbance. This phenomenon is likely due to progressive loss of one of the key enzymes involved in the biosynthetic pathway for dopamine in the basal ganglia: aromatic L-amino acid decarboxylase (AADC). We have developed a gene therapy based on an adeno-associated virus encoding human AADC (AAV2-hAADC) infused into the Parkinsonian striatum. Although no adverse clinical effects of the AAV2-hAADC gene therapy have been observed so far, the ability to more precisely regulate transgene expression or transgene product activity could be an important long-term safety feature. The present study was designed to define pharmacological regulation of the functional activity of AAV2-hAADC transgene product by manipulating L-DOPA and carbidopa (AADC inhibitor) administration in hemi-parkinsonian rats. Thirty days after unilateral striatal infusion of AAV2-hAADC, animals displayed circling behavior and acceleration of dopamine metabolism in the lesioned striatum after administration of a low dose of L-DOPA (5 mg/kg) co-administered with 1.25 mg/kg of carbidopa. This phenomenon was not observed in control AAV2-GFP-treated rats. Withdrawal of carbidopa from a daily L-DOPA regimen decreased the peripheral L-DOPA pool, resulting in almost total loss of L-DOPA-induced behavioral response in AAV2-hAADC rats and a significant decline in striatal dopamine turnover. The serum L-DOPA level correlated with the magnitude of circling behavior in AAV2-hAADC rats. Additionally, AADC activity in homogenates of lesioned striata transduced by AAV2-AADC was 10-fold higher when compared with AAV2-GFP-treated control striata, confirming functional transduction. Our data suggests that the pharmacological regulation of circulating L-DOPA might be effective in the controlling of function of AAV2-hAADC transgene product in PD gene therapy.

There Must Be a Way Out of Here: Identifying a Safe and Efficient Combination of Promoter, Transgene, and Vector Backbone for Gene Therapy of Neurological Disease

There Must Be a Way Out of Here: Identifying a Safe and Efficient Combination of Promoter, Transgene, and Vector Backbone for Gene Therapy of Neurological Disease

AAV9-mediated Expression of a Non-self Protein in Nonhuman Primate Central Nervous System Triggers Widespread Neuroinflammation Driven by Antigen-presenting Cell Transduction

Many studies have demonstrated that adeno-associated virus serotype 9 (AAV9) transduces astrocytes and neurons when infused into rat or nonhuman primate (NHP) brain. We previously showed in rats that transduction of antigen-presenting cells (APC) by AAV9 encoding a foreign protein triggered a full neurotoxic immune response. Accordingly, we asked whether this phenomenon occurred in NHP. We performed parenchymal or intrathecal infusion of AAV9 encoding green fluorescent protein (GFP), a non-self protein derived from jellyfish, or human aromatic L-amino acid decarboxylase (hAADC), a self-protein, in separate NHP. Animals receiving AAV9-GFP into cisterna magna (CM) became ataxic, indicating cerebellar pathology, whereas AAV9-hAADC animals remained healthy. In transduced regions, AAV9-GFP elicited inflammation associated with early activation of astrocytic and microglial cells, along with upregulation of major histocompatibility complex class II (MHC-II) in glia. In addition, we found Purkinje neurons lacking calbindin after AAV9-GFP but not after AAV9-hAADC delivery. Our results demonstrate that AAV9-mediated expression of a foreign-protein, but not self-recognized protein, triggers complete immune responses in NHP regardless of the route of administration. Our results warrant caution when contemplating use of serotypes that can transduce APC if the transgene is not syngeneic with the host. This finding has the potential to complicate preclinical toxicology studies in which such vectors encoding human cDNA's are tested in animals.

Treatment of Congenital Neurotransmitter Deficiencies by Intracerebral Ventricular Injection of an Adeno-Associated Virus Serotype 9 Vector

Dopamine and serotonin are produced by distinct groups of neurons in the brain, and gene therapies other than direct injection have not been attempted to correct congenital deficiencies in such neurotransmitters. In this study, we performed gene therapy to treat knock-in mice with dopamine and serotonin deficiencies caused by a mutation in the aromatic L-amino acid decarboxylase (AADC) gene (Ddc(KI) mice). Intracerebral ventricular injection of neonatal mice with an adeno-associated virus (AAV) serotype 9 (AAV9) vector expressing the human AADC gene (AAV9-hAADC) resulted in widespread AADC expression in the brain. Without treatment, 4-week-old Ddc(KI) mice exhibited whole-brain homogenate dopamine and serotonin levels of 25% and 15% of normal, respectively. After gene therapy, the levels rose to 100% and 40% of normal, respectively. The gene therapy improved the growth rate and survival of Ddc(KI) mice and normalized their hindlimb clasping and cardiovascular dysfunctions. The behavioral abnormalities of the Ddc(KI) mice were partially corrected, and the treated Ddc(KI) mice were slightly more active than normal mice. No immune reactions resulted from the treatment. Therefore, a congenital neurotransmitter deficiency can be treated safely through inducing widespread expression of the deficient gene in neonatal mice.

Safety and tolerability of MRI-guided infusion of AAV2-hAADC into the mid-brain of nonhuman primate

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare, autosomal-recessive neurological disorder caused by mutations in the DDC gene that leads to an inability to synthesize catecholamines and serotonin. As a result, patients suffer compromised development, particularly in motor function. A recent gene replacement clinical trial explored putaminal delivery of recombinant adeno-associated virus serotype 2 vector encoding human AADC (AAV2-hAADC) in AADC-deficient children. Unfortunately, patients presented only modest amelioration of motor symptoms, which authors acknowledged could be due to insufficient transduction of putamen. We hypothesize that, with the development of a highly accurate MRI-guided cannula placement technology, a more effective approach might be to target the affected mid-brain neurons directly. Transduction of AADC-deficient dopaminergic neurons in the substantia nigra and ventral tegmental area with locally infused AAV2-hAADC would be expected to lead to restoration of normal dopamine levels in affected children. The objective of this study was to assess the long-term safety and tolerability of bilateral AAV2-hAADC MRI-guided pressurized infusion into the mid-brain of nonhuman primates. Animals received either vehicle, low or high AAV2-hAADC vector dose and were euthanized 1, 3, or 9 months after surgery. Our data indicate that effective mid-brain transduction was achieved without untoward effects.

Cerebral Infusion of AAV9 Vector-encoding Non-self Proteins Can Elicit Cell-mediated Immune Responses

There is considerable interest in the use of adeno-associated virus serotype 9 (AAV9) for neurological gene therapy partly because of its ability to cross the blood-brain barrier to transduce astrocytes and neurons. This raises the possibility that AAV9 might also transduce antigen-presenting cells (APC) in the brain and provoke an adaptive immune response. We tested this hypothesis by infusing AAV9 vectors encoding foreign antigens, namely human aromatic L-amino acid decarboxylase (hAADC) and green fluorescent protein (GFP), into rat brain parenchyma. Over ensuing weeks, both vectors elicited a prominent inflammation in transduced brain regions associated with upregulation of MHC II in glia and associated lymphocytic infiltration. Transduction of either thalamus or striatum with AAV9-hAADC evinced a significant loss of neurons and induction of anti-hAADC antibodies. We conclude that AAV9 transduces APC in the brain and, depending on the immunogenicity of the transgene, can provoke a full immune response that mediates significant brain pathology. We emphasize, however, that these observations do not preclude the use of AAV serotypes that can transduce APC. However, it does potentially complicate preclinical toxicology studies in which non-self proteins are expressed at a level sufficient to trigger cell-mediated and humoral immune responses.

Long-Term Evaluation of a Phase 1 Study of AADC Gene Therapy for Parkinson's Disease

We report the results of a long-term follow-up of subjects in a phase 1 study of AAV2-hAADC (adeno-associated virus type 2-human aromatic L-amino acid decarboxylase) gene therapy for the treatment of Parkinson's disease (PD). Ten patients with moderately advanced PD received bilateral putaminal infusions of either a low or a high dose of AAV2-hAADC vector. An annual positron emission tomography (PET) imaging with [(18)F]fluoro-L-m-tyrosine tracer was used for evaluation of AADC expression, and a standard clinical rating scale [Unified Parkinson's Disease Rating Scale (UPDRS)] was used to assess effect. Our previous analysis of the 6-month data suggested that this treatment was acutely safe and well tolerated. We found that the elevated PET signal observed in the first 12 months persisted over 4 years in both dose groups. A significantly increased PET value compared with the presurgery baseline was maintained over the 4-year monitoring period. The UPDRS in all patients off medication for 12 hr improved in the first 12 months, but displayed a slow deterioration in subsequent years. This analysis demonstrates that apparent efficacy continues through later years with an acceptable safety profile. These data indicate stable transgene expression over 4 years after vector delivery and continued safety, but emphasize the need for a controlled efficacy trial and the use of a higher vector dose.

Safety and Tolerability of Magnetic Resonance Imaging-Guided Convection-Enhanced Delivery of AAV2-hAADC with a Novel Delivery Platform in Nonhuman Primate Striatum

Degeneration of nigrostriatal neurons in Parkinson's disease (PD) causes progressive loss of aromatic l-amino acid decarboxylase (AADC), the enzyme that converts levodopa (l-DOPA) into dopamine in the striatum. Because loss of this enzyme appears to be a major driver of progressive impairment of response to the mainstay drug, l-DOPA, one promising approach has been to use gene therapy to restore AADC activity in the human putamen and thereby restore normal l-DOPA response in patients with PD. An open-label phase I clinical trial of this approach in patients with PD provided encouraging signs of improvement in Unified Parkinson's Disease Rating Scale scores and reductions in antiparkinsonian medications. However, such improvement was modest compared with the results previously reported in parkinsonian rhesus macaques. The reason for this discrepancy may have been that the relatively small volume of vector infused in the clinical study restricted the distribution of AADC expression, such that only about 20% of the postcommissural putamen was covered, as revealed by l-[3-(18)F]-α-methyltyrosine-positron emission tomography. To achieve more quantitative distribution of vector, we have developed a visual guidance system for parenchymal infusion of AAV2. The purpose of the present study was to evaluate the combined magnetic resonance imaging-guided delivery system with AAV2-hAADC under conditions that approximate the intended clinical protocol. Our data indicate that this approach directed accurate cannula placement and effective vector distribution without inducing any untoward effects in nonhuman primates infused with a high dose of AAV2-hAADC.

Does the aromatic l-amino acid decarboxylase contribute to thyronamine biosynthesis?

Thyronamines (TAM), recently described endogenous signaling molecules, exert metabolic and pharmacological actions partly opposing those of the thyromimetic hormone T3. TAM biosynthesis from thyroid hormone (TH) precursors requires decarboxylation of the l-alanine side chain and several deiodination steps to convert e.g. l-thyroxine (T4) into the most potent 3-T1AM. Aromatic l-amino acid decarboxylase (AADC) was proposed to mediate TAM biosynthesis via decarboxylation of TH. This hypothesis was tested by incubating recombinant human AADC, which actively catalyzes dopamine production from DOPA, with several TH. Under all reaction conditions tested, AADC failed to catalyze TH decarboxylation, thus challenging the initial hypothesis. These in vitro observations are supported by detection of 3-T1AM in plasma of patients with AADC-deficiency at levels (46±18nM, n=4) similar to those of healthy controls. Therefore, we propose that the enzymatic decarboxylation needed to form TAM from TH is catalyzed by another unique, perhaps TH-specific, decarboxylase.

T2 Imaging in Monitoring of Intraparenchymal Real-Time Convection-Enhanced Delivery

Real-time convection-enhanced delivery (RCD) of adeno-associated viral vectors by co-infusion of gadoteridol allows T1 magnetic resonance imaging (T1 MRI) prediction of areas of subsequent gene expression. The use of T2 MRI in RCD is less developed. In addition, the effect of flushing a dead-space volume on subsequent distribution of a therapeutic agent is not known. The value of T2 MRI in RCD was investigated by comparing distribution volumes of saline with immediately after T1 RCD of gadoteridol and by comparing T2, T1, and transgene distribution patterns after viral vector RCD. Adult nonhuman primates underwent saline infusion/T2 acquisition, immediately followed by gadoteridol infusion/T1 acquisition in the putamen and brainstem. Distribution volumes and spatial patterns were analyzed. Gadoteridol and adeno-associated virus encoding human aromatic l-amino acid decarboxylase (AAV2-hAADC) were co-infused under alternating T2/T1 acquisition in the thalamus, and hyperintense areas were compared with areas of subsequent transgene expression. Ratios of distribution volume to infusion volume were similar between saline and gadoteridol RCD. Spatial overlap correlated well between T2 and T1 images. The second infusate followed a spatiotemporal pattern similar to that of the first, filling the target area before developing extra-target distribution. Areas of human L-amino acid decarboxylase expression correlated well with areas of both T1 and T2 hyperintensity observed during RCD. Accuracy of cannula placement and initial infusate distribution may be safely determined by saline infusion without significantly altering the subsequent distribution of the tracer agent. T2 RCD provides detection of intraparenchymal convection- enhanced delivery in the uninjured brain and may predict subsequent distribution of a transgene after viral vector infusion.

Qualitative Imaging of Adeno-Associated Virus Serotype 2–Human Aromatic L-Amino Acid Decarboxylase Gene Therapy in a Phase I Study for the Treatment of Parkinson Disease

Putaminal convection-enhanced delivery (CED) of an adeno-associated virus serotype 2 (AAV2) vector, containing the human aromatic L-amino acid decarboxylase (hAADC) gene for the treatment of Parkinson disease (PD), has completed a phase I clinical trial. To retrospectively analyze magnetic resonance imaging (MRI) and positron emission tomography (PET) data from the phase I trial, correlate those data with similar nonhuman primate (NHP) data, and present how such information may improve future PD gene therapy trials in preparation for the initiation of the phase II trial. Ten patients with PD had been treated with bilateral MRI-guided putaminal infusions of AAV2-hAADC. MRI and PET scans were obtained at baseline (before vector administration) and at various intervals after treatment. Three normal adult NHPs received similar infusions into the thalamus. Imaging studies for both groups are presented, as well as hAADC immunohistochemistry for the NHPs. Early post-CED MRI confirmed the stereotactic targeting accuracy and revealed T2 hyperintensity around the distal cannula tracts, best seen within 4 hours of surgery. Coregistration of post-CED MRI and PET scans revealed increased PET uptake at the sites of T2 hyperintensity. Similar T2 hyperintensities in NHP MRI correlated with hAADC immunohistochemistry. Our analysis confirms the correct targeting of the CED cannula tracts within the target human putamen. Coregistration of MRI and PET confirms colocalization of T2 hyperintensities and increased PET uptake around the distal cannula tracts. Because PET uptake closely correlates with hAADC transgene expression and NHP data confirm this relationship between T2 hyperintensity and hAADC immunohistochemistry, we believe that T2-weighted MRI allows visualization of a significant part of the distribution volume of the hAADC gene therapy. Recommendations for future protocols based on these data are presented.

Real-time MR Imaging With Gadoteridol Predicts Distribution of Transgenes After Convection-enhanced Delivery of AAV2 Vectors

Gene therapies that utilize convention-enhanced delivery (CED) will require close monitoring of vector infusion in real time and accurate prediction of drug distribution. The magnetic resonance imaging (MRI) contrast agent, Gadoteridol (Gd), was used to monitor CED infusion and to predict the expression pattern of glial cell line-derived neurotrophic factor (GDNF) protein after administration of adeno-associated virus type 2 (AAV2) vector encoding human pre-pro-GDNF complementary DNA. The nonhuman primate (NHP) thalamus was utilized for modeling infusion to allow delivery of volumes more relevant to planned human studies. AAV2 encoding human aromatic L-amino acid decarboxylase (AADC) was coinfused with AAV2-GDNF/Gd to confirm regions of AAV2 transduction versus extracellular GDNF diffusion. There was a close correlation between Gd distribution and GDNF or AADC expression, and the ratios of expression areas of GDNF or AADC versus Gd were both close to 1. Our data support the use of Gd and MRI to monitor AAV2 infusion via CED and to predict the distribution of GDNF protein after AAV2-GDNF administration.