Primary outcome measures assessed safety and biomarker evidence of increased brain AADC activity. Safety of the procedure was evaluated by brain MRI 48 hours post-surgery, caregiver report of symptoms at each study visit, neurologist rating of post-surgery involuntary movements (dyskinesia) at each study visit, and caregiver diary of sleep and behavior symptoms at selected visits. Evidence of biological AADC activity was measured by [18F]FDOPA PET and analysis of CSF neurotransmitter metabolites before and after surgery. Secondary outcome measures assessed clinical efficacy as expressed by improvements in symptoms and motor function. Caregivers kept a detailed log of the duration and severity of all oculogyric crises (OGC Log) before and after surgery. A checklist of AADC deficiency-related symptoms was reviewed at each study visit. Caregivers periodically recorded sleep and behavior observations in a 7-day diary. Gross motor function was evaluated using the Gross Motor Function Measure-88 (GMFM-88). All measures are described in further detail below, grouped by category (clinical/laboratory/imaging).
A systematic assessment of adverse events and side effects was performed at each visit with the study neurologist (Screening, Baseline, Weeks 1, 2 ,4, 5, 6, 7, 8, and Months 3,6,12,18 and 24). Information was recorded on an Adverse Events (AE) Log which included type of AE, dates of onset and resolution, severity, and perceived relationship to experimental therapy (Table 4). The severity of each AE was rated based on the NCI Common Terminology Criteria for Adverse Events v4.0 (CTCAE; https://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/CTCAE_4.03.xlsx). A general medical and a full neurologic examination was conducted at each study visit with selected examinations recorded on video.
At each study visit with the neurologist (see timepoints above), a standard checklist of AADC deficiency-related symptoms was reviewed, in which parents were asked to judge the severity of each symptom as “major” (frequent and/or severe), “minor” (infrequent and/or mild), or absent. Subjects were also examined by the neurologist at each visit to assess the severity of involuntary movements, or dyskinesia. A dyskinesia score, based on the Abnormal Involuntary Movement Scale (AIMS) was calculated by assessing the severity of involuntary movements in each of 7 body regions on a scale of 0 (none) to 4 (severe), for a maximum possible score of 28 (Fig. 5).
Caregivers documented the duration and severity of all OGC that occurred during the study period, beginning 2 months prior to surgery, in an OGC Log. OGCs typically occur 2–3 times per week and each episode lasts from 30 minutes to 8 hours; therefore, the average weekly duration and severity of episodes can be accurately and reliably quantified. We created a 3-point scale to describe episode severity based on clinical experience: Grade 1 (mild) = eye deviation only; Grade 2 (moderate) = eye deviation associated with involuntary facial movements; Grade 3 (severe) = eye deviation associated with dyskinesia or dystonia of the trunk and/or limbs. Parents were trained at the initial screening visit to complete the OGC log by documenting a start time, end time, and severity score (1–3) for each episode. They then prospectively recorded all observed episodes throughout the study period. An OGC severity score, consisting of a product of the duration (hours) and severity (grade 1–3) was calculated by summing the number of hours of each severity category per calendar month, and then calculating the weekly (7-day) average score for that month. For example, a score of 15 may represent 5 hours per week of severe (severity score 3) symptoms, 15 hours per week of mild (severity score 1) symptoms, or an intermediate number of hours of symptoms of mixed severity.
A standardized behavior and sleep diary was used to capture sleep and mood dysfunction resulting from brain dopamine dysregulation. Subject’s caregivers were trained in the completion of a symptom diary by study center personnel. The symptom diary was completed twice during the period between the Screening and Baseline visits, each over a consecutive 7-day period at least 1 month apart. Follow-up observations for 7-day periods were recorded at Week 6 and Months 3, 6, 12, 18 and 24 after surgery.
Gross motor function was evaluated using the GMFM-88. The total score was derived as an unweighted average of the 5 dimension scores: lying and rolling (17 items); sitting (20 items); crawling and kneeling (14 items); standing (13 items) and walking, running, and jumping (24 items). Each dimension score was defined as percentage of maximum score for the dimension in question, and the maximum possible total score was 100%. The evaluations were performed by a pediatric physical therapist and recorded on video at Baseline, 3, 6 12, 18 and 24 months after gene delivery. The assessments were then independently scored by two therapists, the one who had performed the test in-person, and a second therapist who reviewed each recorded session on video (post-hoc, blinded to the timepoint of each assessment). When the total scores of the 2 independent assessments were within 2 percentage points of each other, the score of the original (in-person) assessor was retained. When there was a difference of greater than 2 points between the total scores, a third reviewer (TSP) reviewed the video to determine each individual item’s final score and ensure accurate and consistent scoring across subjects.
The parents of all the patients participated in a formal survey interview using the Vineland Adaptive Behavior Scale, Second Edition (VABS-II), a measure of adaptive behavior and developmental functioning for individuals from birth through 90 years of age31. This tool is administered to the primary caregiver of an individual being assessed and provides information in various domains of functioning. Specifically, there are four domains (Communication, Daily Living Skills, Socialization, and Motor Skills), up to 11 subscales (three each for all domains except Motor, which has two), and an overall Adaptive Behavior Composite Score. Standard scores (population mean = 100, SD = 15) are produced for the domain and composite scores, scaled scores (called v-scale scores, with a population mean = 15, SD = 3) are produced for the subscale scores, age equivalents can also be generated and may be combined to determine an overall Adaptive Behavior Composite score. Strong internal consistency, test-retest reliability, and validity have consistently been demonstrated. However, due to the significant global developmental delays and limited abilities of our subject cohort, there was no utility in calculating formal standard, scaled, and/or composite scores. Instead, raw score comparisons were reviewed across time points to assess any positive developmental progress (Supplementary Table S1). The VABS-II scale was administered at Baseline, 12 and 24 months after surgery.
CSF was collected by lumbar puncture with analysis of the following metabolites: homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), 3-O-methyldopa (3-OMD), and 5-methyltetrahydrofolate (5-MTHF)32. Plasma AADC enzyme activity was determined by quantitative methods33.
MR scans were performed pre-operatively as part of the screening process, during the infusion procedure, and then at 48 hours following the surgical procedure, with a final scan obtained 24 months following the procedure. The 48-hour MR scan used a ‘rapid’ protocol and consisted of a 3D T2 FSE sequence, and axial diffusion.
6-[18F]-fluoro-L-DOPA (FDOPA) is a PET radiopharmaceutical used to image dopamine synthesis capacity. PET scans were performed at screening, and at 3 months, with a final study planned 24 months after the procedure. The pediatric dose of 0.3 mCi for FDOPA was based on 10% of the adult dose of 3 mCi (UCSF RUA approval: RU133031, OSU approval: 2018H0269). PET scans were analyzed for quantification of AADC activity and differences in the distribution of AADC activity between baseline and post-surgery scans.
[123I]ioflupane (DaTscan™) selectively binds to presynaptic dopamine transporters and provides a method for imaging nigrostriatal terminals in the striatum. DaTscan™ is a FDA-approved radiopharmaceutical used in conjunction with single photon emission computed tomography (SPECT) scan for use in adults. A DaTscan was performed at the screening visit to document nigrostriatal pathway integrity.