title: Production of human factor  VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector.
abstract: Hemophilia  A is the most common X-linked bleeding disorder; it is caused by deficiency of  coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from  human cell line is a major goal for treating hemophilia patients. We prepared a  full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral  vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing  a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES,  resulting in a 16.6-kb construct. In parallel, an eGFP control vector was  generated, resulting in a 10.1- kb construct. The 293T cells were transfected  with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K  cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were  4,410 (±931.7)- and 295,400 (±75,769)-fold higher than in virgin cells. In  293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000  (±493,700)- and 308,000 (±139,300)-fold higher than in virgin cells. The amount  of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/μg protein. These data demonstrate  the efficacy of the bicistronic retroviral vector expressing FVIII-FL and  MGMT(P140K), showing that it could be used for producing the FVIII-FL protein  in a human cell line.
authors.affiliation: Hemocentro de Ribeirão Preto, Hospital das Clínicas, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil. fontesam@hemocentro.fmrp.usp.br
authors.forename: A M
authors.initials: AM
authors.lastname: Fontes
authors.forename: F U F
authors.initials: FU
authors.lastname: Melo
authors.forename: L J
authors.initials: LJ
authors.lastname: Greene
authors.forename: V M
authors.initials: VM
authors.lastname: Faça
authors.forename: Y
authors.initials: Y
authors.lastname: Lin
authors.forename: S L
authors.initials: SL
authors.lastname: Gerson
authors.forename: D T
authors.initials: DT
authors.lastname: Covas
mesh_terms: D005169:Factor VIII / Q000096:biosynthesis* / Q000235:genetics; D023061:Gene Order; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D012190:Retroviridae / Q000235:genetics*
chemical_list: D005169:Factor VIII