title: Optimizing Transgene Configuration and Protein Fusions to Maximize Dopamine Production for the Gene Therapy of Parkinson's Disease.
abstract: Pharmacological dopamine replacement therapies provide the most well-established treatments for Parkinson's disease (PD). However, these long-term treatments can lead to motor complications and off-target effects. ProSavin(®), a lentiviral vector (LV)-based gene therapy approach aimed at restoring local and continuous dopamine production, through delivery of three enzymes in the dopamine biosynthesis pathway, was demonstrated to be safe and well-tolerated in a phase I/II clinical study of patients with advanced PD. Although improvements in motor behaviour were observed, the data indicated that higher levels of dopamine replacement might be required to maximize benefit. We attempted to increase production of dopamine, and its precursor L-Dopa in LV-transduced cells, by optimizing the gene order in the ProSavin expression cassette, and by creating fusions of two or three of the transgenes, using linker sequences. In vitro analysis showed that several gene arrangements provided significantly increased dopamine and/or L-Dopa production compared with ProSavin, and that LV titers and transgene expression were not affected by introducing gene fusions. One vector, equine infectious anemia virus (EIAV)-TCiA, was selected for further characterization and showed significant improvements in dopamine and L-Dopa production compared with ProSavin, in human neuronal cells. Further characterization of EIAV-TCiA demonstrated expression of all three dopamine enzymes in vivo and faithful delivery and integration of the expected gene expression cassette within the genome of target cells, as assessed by Northern and Southern blotting. In conclusion, we have developed a novel LV vector with an increased capacity for L-Dopa and dopamine production compared with the current ProSavin vector. Clinical evaluation of this vector will be performed to assess the benefits in patients with PD.
authors.affiliation: 1 Oxford BioMedica (UK) Ltd. , Oxford, United Kingdom.
authors.forename: Hannah J
authors.initials: HJ
authors.lastname: Stewart
authors.affiliation: 1 Oxford BioMedica (UK) Ltd. , Oxford, United Kingdom.
authors.forename: G Scott
authors.initials: GS
authors.lastname: Ralph
authors.affiliation: 2 Henry Wellcome L.I.N.E., University of Bristol , Bristol BS1 3NY, United Kingdom.
authors.forename: Liang
authors.initials: L
authors.lastname: Fong-Wong
authors.affiliation: 2 Henry Wellcome L.I.N.E., University of Bristol , Bristol BS1 3NY, United Kingdom.
authors.forename: Iain
authors.initials: I
authors.lastname: Strickland
authors.affiliation: 1 Oxford BioMedica (UK) Ltd. , Oxford, United Kingdom.
authors.forename: Laura
authors.initials: L
authors.lastname: McCloskey
authors.affiliation: 1 Oxford BioMedica (UK) Ltd. , Oxford, United Kingdom.
authors.forename: Lucy
authors.initials: L
authors.lastname: Barnes
authors.affiliation: 1 Oxford BioMedica (UK) Ltd. , Oxford, United Kingdom.
authors.forename: Ian
authors.initials: I
authors.lastname: Blount
authors.affiliation: 1 Oxford BioMedica (UK) Ltd. , Oxford, United Kingdom.
authors.forename: Owen
authors.initials: O
authors.lastname: Wells
authors.affiliation: 1 Oxford BioMedica (UK) Ltd. , Oxford, United Kingdom.
authors.forename: Christelle J M
authors.initials: CJ
authors.lastname: Truran
authors.affiliation: 1 Oxford BioMedica (UK) Ltd. , Oxford, United Kingdom.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Kingsman
authors.affiliation: 3 AP-HP, Groupe Hospitalier Henri-Mondor, DHU PePsy, Neurochirurgie, INSERM IMRB U955 eq.14, Université Paris Est Créteil (UPEC) , Faculté de Médecine, Creteil, F-94010, France .
authors.forename: Stéphane
authors.initials: S
authors.lastname: Palfi
authors.affiliation: 1 Oxford BioMedica (UK) Ltd. , Oxford, United Kingdom.
authors.forename: Kyriacos A
authors.initials: KA
authors.lastname: Mitrophanous
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004298:Dopamine / Q000096:biosynthesis*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010300:Parkinson Disease / Q000235:genetics / Q000628:therapy*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D004298:Dopamine