Mitochondrial genetic therapy in Parkinson’s disease

• Autor(a) principal: Baptista, Cátia Marlene do Carmo
• Data de Publicação: 2015
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/6459
• topic: Doença de Parkinson; Mutações Mitocondriais; Nanopartículas; Terapia Génica Mitocondrial; Domínio/Área Científica::Engenharia e Tecnologia:: Outras Engenharias e Tecnologias

Mitochondria have their own genome, a circular double-stranded genome that encodes 13 proteins involved in electron transport and oxidative phosphorylation (OXPHOS). They are present in 10–100 copies per somatic cell and within the cell mitochondrial DNA (mtDNA) copies could be identical in sequence (homoplasmy) or wild-type and mutant mtDNA could exist in different ratios (heteroplasmy). Heteroplasmy occurs mainly because mtDNA is particularly susceptible to mutations due its proximity to reactive oxygen species (ROS) generation sites. MtDNA dysfunction is characterized by a defective OXPHOS where ATP demands are not reached. The phenotype of the mitochondrial disease depends on the mtDNA heteroplasmy, when a threshold in the region of 60–90% mutated mtDNAs is reached. The idea that mitochondrial dysfunction could influence Parkinson disease (PD) arose with the discovery of the mechanism of action of MPTP in the early 1980s that affected electron flow in Complex I (CI). Different CI related mutations have been associated with PD and two of them were identified in mtND1 gene. This fact makes the development of a mtND1 construct a very interesting approach for mitochondrial gene therapy (MGT) purposes in PD treatment. Throughout this year, it was developed a nanocarrier to transport a designed vector with possible application in MGT. The vector was constructed using pCAG-GFP plasmid and the mitochondrial gene ND1 and CaCO3 were developed for the directed transport of the plasmid towards mitochondria. This research development was divided into three main stages: i) construction of a vector using an OXPHOS CI gene, mtND1 and the pCAG-GFP plasmid; ii) study of different recombinant hosts to produce the pCAG-GFP-mtND1 vector; and iii) development of a formulation for the efficient pDNA delivery into mitochondria. The mtND1 construct was successfully obtained and inserted into different E. coli strains. The growth profiles were very similar between the strains, being observed sligth differences in the pDNA specific yields. The nanocarries obtained were biocompatible, displaying encapsulation efficiencies (EE) above 50%, sizes below 280 nm and positive zeta potential, making them very suitable for MGT.