title: Production of minigene-containing retroviral vectors using an alphavirus/retrovirus hybrid vector system.
abstract: In an attempt to increase the synthesis of human clotting factors VIII and IX in transduced cells, optimized expression cassettes containing genomic genelike elements (minigenes) were assembled. Plasmid DNA containing factor VIII or factor IX minigenes and driven by three human cellular promoters (albumin, factor IX, PGK) or the strong viral promoter RSV-LTR were electroporated into TE671 and HepG2 cell lines, and clotting factor levels were determined by ELISA. In comparison with a parallel transfection of MLV-LTR-promoted retroviral vector plasmid DNAs, the PGK- and RSV-LTR-promoted minigene constructs produced equal or greater amounts of clotting factor proteins. A factor IX minigene cassette was cloned into the retrovirus-based gene transfer vector LN (in both forward and reverse orientations) and the minigene vector was introduced into the Phoenix retroviral packaging cell line. Analysis of neo(r) cells demonstrated that insertion of a factor IX minigene into the retroviral vector LN resulted in rearrangement of the factor IX sequence and loss of factor IX expression in the Phoenix packaging cell line. The same factor IX minigene was then inserted into an alphavirus/retrovirus hybrid vector that facilitates the synthesis of retroviral vector RNA in the cytoplasm of cells. Alphavirus/retrovirus virions were produced and used to transduce the Phoenix retroviral vector packaging cell line. The cytoplasmically produced factor IX minigene-containing retroviral vectors were collected and used to transduce TE671 cells. Analysis of transduced cells demonstrated stable transfer of the minigene and expression of factor IX.
authors.affiliation: Clinical Gene Therapy Branch/National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-1851, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Wahlfors
authors.forename: R A
authors.initials: RA
authors.lastname: Morgan
mesh_terms: D000524:Alphavirus / Q000235:genetics*; D002460:Cell Line; D018274:Electroporation / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection; D019076:Transgenes
chemical_list: D005169:Factor VIII; D005164:Factor IX