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Platform for generation of a human cell line with high copy number of cDNA relative to synthetic wild type coagulation factor VIIIDB

Grant number: 12/00839-8
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): April 01, 2012
Effective date (End): December 31, 2012
Field of knowledge:Biological Sciences - Genetics
Principal Investigator:Aparecida Maria Fontes
Grantee:Angelo Luis Caron
Home Institution: Hemocentro de Ribeirão Preto. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP (HCMRP). Secretaria da Saúde (São Paulo - Estado). Ribeirão Preto , SP, Brazil

Abstract

Haemophilia A is a genetic X-linked disorder caused by the coagulation factor VIII (FVIII)deficiency. It affects 1 in 5,000 male births and is manifested by spontaneous bleeding or following trauma. The current treatment is the replacement therapy with plasma derived FVIII (pdFVIII) or recombinant FVIII (rFVIII). Despite the advantages of using rFVIII when compared to pdFVIII, as the lowest risk of viral transmission, the recombinant is expensive and has a limited availability. A forthcoming area in science is Synthetic Biology that can provide new biopharmaceutical products in a fast and effective way. The construction of the first prokaryote organism by means of the synthetic Biology guided us to the hypothesis that the same knowledge could be used to construct a functional FVIII. Besides, with the advances in cell culture technologies, that have allowed the use of human cell lines in the production of therapeutic proteins, we choose one of these cell lines to produce FVIII. Also, several clinical trials have shown the efficiency of the lentiviral system that allows multiple cycles of transduction of the cDNA into the cells genome, an important step to obtain several gene copies of interest. In conclusion, the purpose of this project is the development of a platform for the generation of a human cell line with a permanent production of synthetic FVIII. To reach this, the cell line will be submitted through multiple transduction cycles with a third generation lentiviral vector carrying the synthetics wild type FVIIIDB and the neomycin cDNAs. In the first, fifth and tenth cycle of transduction the cell will be characterized as: copy number relative of FVIII in the host cell genome; level of FVIII mRNA and; level of FVIII protein. When finished the transduction cycles the cell will be treated with geneticin to selection of a cell clone with high expression levels of FVIII.