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Title: Functional characterisation of aromatic L-amino acid decarboxylase (AADC) deficiency using a patient-derived dopaminergic cell model
Author: Kramer, Karolin
ISNI:       0000 0004 8507 6265
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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Background: Aromatic L-amino acid decarboxylase (AADC) deficiency is a severe pharmacoresistant neurological disorder due to inherited autosomal recessive loss-of-function mutations in the DDC gene. The resultant impairment of AADC enzyme activity severely impacts on monoamine synthesis, leading to reduced levels of dopamine and serotonin. Affected patients present with marked neurodevelopmental delay, hypotonia, oculogyric crises and autonomic dysfunction. Currently, there are few truly disease-modifying therapies. Aims: To generate AADC patient-derived induced pluripotent stem cells (iPSC) for subsequent differentiation into midbrain dopaminergic (mDA) neurons, and to utilise this model to better define disease mechanisms and test novel therapeutic strategies. Methods: Patient and age-matched control fibroblasts were reprogrammed into iPSC using Sendai Virus methods. A modified dual SMAD inhibition protocol was then utilised for differentiation of all iPSC lines to day 65 of maturation. The generated neuronal model was then analysed for mature mDA neuronal identity and AADC disease-specific features. Results: iPSC lines were generated from skin fibroblasts derived from two patients with AADC deficiency. One patient harboured a homozygous missense mutation (p.R347G) and the other was a compound heterozygote for a nonsense variant (p.Arg7*) and missense mutation (p.C100S) in DDC. For the project two iPSC lines from one age-matched control subject were used that were previously reprogrammed in my host laboratory. Generated iPSC lines were confirmed as being truly pluripotent, then successfully differentiated into midbrain dopaminergic neurons, with characteristic neuronal morphology, expressing tyrosine hydroxylase (TH) and microtubule-associated protein 2 (MAP2). There was no evidence of neurodegeneration in the patient lines. A number of disease-specific features were identified, including significantly marked reduction of AADC enzyme activity and dysregulation of the dopaminergic system in patient mDA neurons when compared to the age-matched control. Preliminary data also shows successful lentiviral rescue of the patient-derived mDA cell model. Conclusion: The iPSC-derived mDA neuronal model represents an ideal platform to further elucidate disease mechanisms, as well as to screen novel pharmacological agents for AADC deficiency.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available