Intraputaminal Gene Delivery in Two Patients with Aromatic L‐Amino Acid Decarboxylase Deficiency

ABSTRACT Background Aromatic l‐amino acid decarboxylase deficiency (AADCD) is a rare, early‐onset, dyskinetic encephalopathy mostly reflecting a defective synthesis of brain dopamine and serotonin. Intracerebral gene delivery (GD) provided a significant improvement among AADCD patients (mean age, ≤6 years). Objective We describe the clinical, biological, and imaging evolution of two AADCD patients ages >10 years after GD. Methods Eladocagene exuparvovec, a recombinant adeno‐associated virus containing the human complimentary DNA encoding the AADC enzyme, was administered into bilateral putamen by stereotactic surgery. Results Eighteen months after GD, patients showed improvement in motor, cognitive and behavioral function, and in quality of life. Cerebral l‐6‐[18F] fluoro‐3, 4‐dihydroxyphenylalanine uptake was increased at 1 month, persisting at 1 year compared to baseline. Conclusion Two patients with a severe form of AADCD had an objective motor and non‐motor benefit from eladocagene exuparvovec injection even when treated after the age of 10 years, as in the seminal study.


ABSTRACT: Background
Background: Aromatic L-amino acid decarboxylase deficiency (AADCD) is a rare, early-onset, dyskinetic encephalopathy mostly reflecting a defective synthesis of brain dopamine and serotonin. Intracerebral gene delivery (GD) provided a significant improvement among AADCD patients (mean age, ≤6 years). Objective Objective: We describe the clinical, biological, and imaging evolution of two AADCD patients ages >10 years after GD. Methods Methods: Eladocagene exuparvovec, a recombinant adeno-associated virus containing the human complimentary DNA encoding the AADC enzyme, was administered into bilateral putamen by stereotactic surgery.

Results
Results: Eighteen months after GD, patients showed improvement in motor, cognitive and behavioral function, and in quality of life. Cerebral L-6-[ 18 F] fluoro-3, 4-dihydroxyphenylalanine uptake was increased at 1 month, persisting at 1 year compared to baseline. Conclusion Conclusion: Two patients with a severe form of AADCD had an objective motor and non-motor benefit from eladocagene exuparvovec injection even when treated after the age of 10 years, as in the seminal study.
Aromatic L-amino acid decarboxylase deficiency (AADC) deficiency (AADCD; OMIM 608643) is a rare autosomal recessive disease because of pathogenic variants of the dopa decarboxylase (DDC gene); subsequent monoamine defect leads to early-onset disorder characterized by hypotonia, movement disorders, dysautonomic features, and behavioral disturbances. [1][2][3][4][5] Medications are disappointing, and 80% of the patients suffer from severe motor and cognitive impairment and risk of premature death. 3,6 Gene delivery (GD) for AADCD provides the defective gene by injecting a recombinant adeno-associated virus 1  containing the human complimentary DNA encoding the DDC gene, such as eladocagene exuparvovec, directly into the brain to increase striatal synthesis of dopamine. 7 Thirty-nine young patients with AADCD (mean age, ≤6 years old) have benefited from intraputaminal GD, [8][9][10][11] and seven from midbrain GD, 12 with significant improvement in motor and non-motor functions. Here, we report the 18-month follow-up of the first two patients >10 years old treated in Europe by intraputaminal eladocagene exuparvovec delivery.

Selection of the Patients
Patients were referred for GD by their local pediatric neurologist and were selected as candidates for eladocagene exuparvovec injection based on their symptomatic presentation, lack of improvement on traditional standard of care, and clinical suitability for GD by a multidisciplinary multicentric group of experts. Eladocagene exuparvovec was available under a temporary authorization for compassionate use, according to the article L.5121-12 of the Public Health Code, in Montpellier University Hospital. Baseline and post-GD assessment schedule of the patients and neurosurgical procedure according to the fabricant recommendations have been validated by the French Drug Agency as daily care including expenses support.

Surgical Procedure
Each patient received a total of 1.8 Â 10 11 viral genomes delivered as four intraputaminal injections of 80 μL conducted at a rate of 3 μL/min. Protocols for surgery, pharmacy, storage, and shipment of eladocagene exuparvovec have been previously described. 7,13 Clinical and Paraclinical Assessment Clinical data were collected in the medical file and at each follow-up visit, specifically to motor function (Gross Motor Function Measure-88 Item, Manual Ability Classification System), cognitive function (Vineland Adaptive Behavior Scales), sleep and mood disturbances, irritability, comfort, wellbeing, and ease of caregiving according to Caregiver Priorities and Child Health Index of Life with Disabilities (CPCHILD) questionnaire, gastrointestinal and feeding difficulties, weight, respiratory infections, pharmacological changes, dyskinesia, and oculogyric crisis (Pearson's score). 12 Brain magnetic resonance imaging, L-6-[ 18 F] fluoro-3, 4-dihydroxyphenylalanine ( 18 F-DOPA) positron emission tomography (PET) were performed before GD and 1 month and 1 year after GD. Cerebrospinal fluid analysis (cell count, biochemical analysis, and neurotransmitters' profile) was performed before GD and 18 months after GD. Anti-adeno-associated virus type 2 titers were monitored at baseline and at 3, 6, 12, and 18 months after GD.

Results
Patient 1 and 2 benefited from GD (1.8 Â 10 11 viral genomes delivered as four intraputaminal injections) at ages 10.7 years and 11.4 years, respectively. Past medical history is summarized in Table S1. Before GD, patients had no head control and limited voluntary movement despite treatment strategy conducted according to the guidelines (Videos 1, 4). 3 Longitudinal assessment data are summarized in Table 1.

Motor Function
Both patients exhibited improved motor function, especially axial tone and voluntary movements of limbs. Patient 1 could control the head at 10 months, and patient 2 at 6 months. Spontaneous and voluntary movements dramatically improved from 2 months after GD. Patient 1 could reach an object at 6 months, whereas patient 2 could grab from 12 months (Videos 2, 3, 5, 6). Dystonia and oculogyric crisis (OGC) (duration, frequency, and severity) diminished progressively from 3 months after GD.

Behavioral and Cognitive Function
At baseline, both patients had mood and sleep disturbances, excessive crying, and could only communicate through eye contact and grunting. From the fourth week after GD, caregivers/ families reported improvement of sleep disturbances, irritability, and wakefulness. Joyful behavior, increased alertness, and better communication were also reported for both patients, including patient 1's newly gained ability to laugh from month 6 after GD (Table 1, CP child, section 0). At baseline, patient 1 could only grunt, and patient 2 could not produce any sound. Six months after GD, both patients could emit various sounds and syllables used to call the caregivers; 18 months after GD, patient 1 could emit bisyllabic words. Improvement of cognitive subdomains is highlighted by Vineland subscore (Table 1, Vineland scores).

Other Functions
Feeding abilities (including weaning from tube feeding in patient 2) resulted in increased in body weight of both patients; respiratory infections in patient 2 significantly improved (Table 1).

Adverse Events
No serious adverse events were reported during the surgical procedure or follow-up. Patient 2, with a known, well-controlled epilepsy, exhibited a cluster of brief focal seizure during a noninfectious febrile peak 36 h after GD. Moderate and transient dyskinesia occurred in both patients from 4 weeks following

Paraclinical Assessment
Post-surgery magnetic resonance imaging did not show any feature of bleeding or infection. Both patients showed severe impairment of 18 F-DOPA uptake at baseline, with improvement in uptake at 1 month persisting at 1 year compared with baseline, albeit more heterogeneously in patient 2 ( Fig. 1 and Table S2). There was an increased CSF level the dopamine metabolite homovanillic acid (HVA), but not of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) level in patient 2 at last follow-up compared to baseline. Dopamine levels remained stable in patient 1 (Table 1).

Discussion
We report the first European compassionate experience of intraputaminal delivery of eladocagene exuparvovec in two children >10 years old with a severe form of AADCD. This welltolerated treatment resulted in consistent improvement of motor and non-motor function. Follow-up was characterized by early improvement in sleep and mood disturbances, alertness, and communication skills, especially expression abilities. Both patients managed to hold up their head before 8 months. Spontaneous movements were dramatically enriched, allowing the patients to reach/grab objects; increased Gross Motor Function Measure-88 Video Item score highlight motor outcome. This progress, as well as resolution of OGC, and ability to feed orally resulted in remarkable improvement of quality of life. Previous studies showed faster and greater progress in young patients, especially regarding gross motor progress and fine motor skills (ability to reach/grab or eat with hands), 7-9,11,12 with an even better functional recovery in patients young than 3; 8 data concerning patients >10 years old are limited. Indeed, Kojima et al 9 reported four patients >10 years old with improvement of cognitive and motor functions (head control and sitting with support: all patients before 8 months after GD; stand with support: 3 of 4 patients before 18 months after GD; aided walk: 1 patient; all patients could grip at the last follow-up) and disappearance of OCG and dystonia following intraputaminal administration of rAAV-hAADC gene therapy. 9 Side effects were limited, dyskinesia occurred as reported in other studies; 7-9,11,12 they lasted less than 6 months and were easily controlled.
Because 18 F-DOPA is rapidly washed out from putaminal neurons if not metabolized into dopamine, evidence of recovery of a certain level of cerebral AADC activity is provided by recovery of putaminal uptake of 18 F-DOPA for both patients 1 year after GD, albeit a partial decrease in uptake compared with uptake 1 month after GD for patient 2. These results are similar to those of Hwu et al, 7 confirming the importance of 18 F-DOPA PET in providing indication of the biological effectiveness of GD.
Restoration of AADC activity is also supported by increased CSF levels of HVA in patient 2. Patient 1's HVA CSF dosage was not increased from baseline to last follow-up. Although a strong correlation has been reported between the post-treatment HVA level and motor outcome, 11 limited changes in HVA levels have been encountered in previously reported patients with significant clinical improvement. 8,12 Moreover, CSF dopamine metabolites levels after intracerebral DDC gene delivery have not been assessed in animal models, therefore, scientific experimental data concerning CSF dopamine metabolites as reliable biomarkers of AADC activity restauration after gene delivery are lacking. The value of CSF biomarkers as a relevant clue for AADC activity restauration and/or prognosis indicator of good motor outcome will need to be further studied with standardized timepoints of assessement.
As in previous series of AADCD patients who received gene delivery, clinical assessment of the two patients was unblinded, and part of evaluation was based on caregivers rating (especially wakefulness, behavior, and sleep); 7-9,11,12 this approach intrinsically includes bias related to the magnitude of expected benefit from the caregivers and clinicians' awareness of the patient's status. Moreover, various and heterogeneous scales or scores have been used among the series, 7-9,11,12 resulting in limitations in the attempts to compare patient's outcome, especially concerning functional improvement, which probably represent one of the most meaningful and significant objective of therapy in such severe disease.

Conclusion
Robust evidence corroborates the absence of the degenerative process in AADC. 3,14,15 This basic rationale supports extending gene therapy to patients older than those studied in the pivotal clinical trials. The clinical outcome in our two patients is concordant with previous reports and confirms that intraputaminal DDC gene delivery has a consistent safe profile and can be considered for use in teenagers or older patients suffering from a severe form of AADCD with significant impairments. Unified and standardized monitoring of the patients will aid in better understanding of the biological and clinical effect of this innovative therapeutic procedure.

Disclosures
Ethical Compliance Statement: The authors confirm that the approval of an institutional review board was not required for this work. All legal representative of the patients provided written and oral informed consent according to the Declaration of Helsinki to perform the procedure, collect and share the data including videos. All authors confirm that they have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.

Supporting Information
Supporting information may be found in the online version of this article. Table S1. Characteristics and past medical history of patients. Table S2. Quantification of the specific putaminal uptake at baseline, 1 month, and 1 year after GD.