New cell lines for the manufacture of lentivirus

• Autor(a) principal: Costa, Diogo Lima de Vilhena Magalhães
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
• Texto Completo: http://hdl.handle.net/10400.14/16121
• topic: Gene therapy; Large T antigen; Lentivirus; Retrovirus; Cell line; Linha celular; Terapia génica

Background: Gene therapy consists on medical treatment that aims to modulate an individual’s gene expression. To this end, many different gene transfer vehicles called vectors have been developed, including virus derived vectors. Showing promising characteristics, lentiviral vectors still have problems associated to them, especially in their production process, limited by low titers. To increase viral vector titer and producer cell growth, oncogenes such as SV40 Large T (T-Ag) antigen are expressed in producer cell lines, which decrease the safety of the vector preparations. Objectives: With the objective to find alternative cell substrates to HEK293T suitable for high titer vector production, three non-human cell lines were transformed with T-Ag oncogene and transfected with a lentiviral construct. Their vector production and transfection efficiency was characterized. The strength of several promoters to drive the expression of viral components was also evaluated in these cell lines. Results and conclusions: CAG and CMV revealed to be the most promising, although CAG delivers lower titers. In this work, it was shown that Age1.CR and Vero cell lines have the potential to deliver enhanced lentivector titers when expressing T-Ag, which conferred higher transfection efficiencies. Also, HEK293 cells expressing T-Ag were compared to their parental cell line in terms of cell growth and glycolysis in an attempt to understand cellular alterations induced by the oncogene. The results herein obtained will contribute to the development of stable lentivector producer cell lines and for the further understanding of the T-Ag’s influence in virus production.