title: Extensive Replication of a Retroviral Replicating Vector Can Expand the A Bulge in the Encephalomyocarditis Virus Internal Ribosome Entry Site and Change Translation Efficiency of the Downstream Transgene.
abstract: We have developed retroviral replicating vectors (RRV) derived from Moloney murine gammaretrovirus with an amphotropic envelope and an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-transgene cassette downstream of the env gene. During long-term (180 days) replication of the vector in animals, a bulge of 7 adenosine residues (A's) in the J-K bifurcation domain sometimes serially added A's. Therefore, vectors with 4-12 A's in the A bulge in the J-K bifurcation domain were generated, and the impact of the variants on transgene protein expression, vector stability, and IRES sequence upon multiple infection cycles was assessed in RRV encoding yeast-derived cytosine deaminase and green fluorescent protein in vitro. For transgene protein expression, after multiple infection cycles, RRV-IRES with 5-7 A's gave roughly comparable levels, 4 and 8 A's were within about 4-5-fold of the 6 A's, whereas 10 and 12 A's were marked lower. In terms of stability, after 10 infection cycles, expansion of A's appeared to be a more frequent event affecting transgene protein expression than viral genome deletions or rearrangement: 4 and 5 A's appeared completely stable; 6, 7, and particularly 8 A's showed some level of expansion in the A bulge; 10 and 12 A's underwent both expansion and transgene deletion. The strong relative translational activity of the 5 A's in the EMCV IRES has not been reported previously. The 5A RRV-IRES may have utility for preclinical and clinical applications where extended replication is required.
authors.affiliation: 1 Tocagen Inc. , San Diego, California.
authors.forename: Amy H
authors.initials: AH
authors.lastname: Lin
authors.affiliation: 1 Tocagen Inc. , San Diego, California.
authors.forename: Yanzheng
authors.initials: Y
authors.lastname: Liu
authors.affiliation: 1 Tocagen Inc. , San Diego, California.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Burrascano
authors.affiliation: 1 Tocagen Inc. , San Diego, California.
authors.forename: Kathrina
authors.initials: K
authors.lastname: Cunanan
authors.affiliation: 2 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California-Los Angeles , Los Angeles, California.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Logg
authors.affiliation: 1 Tocagen Inc. , San Diego, California.
authors.forename: Joan M
authors.initials: JM
authors.lastname: Robbins
authors.affiliation: 2 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California-Los Angeles , Los Angeles, California.|3 Department of Cell Biology, Miller School of Medicine, University of Miami , Miami, Florida.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Kasahara
authors.affiliation: 1 Tocagen Inc. , San Diego, California.
authors.forename: Harry
authors.initials: H
authors.lastname: Gruber
authors.affiliation: 1 Tocagen Inc. , San Diego, California.
authors.forename: Carlos
authors.initials: C
authors.lastname: Ibañez
authors.affiliation: 1 Tocagen Inc. , San Diego, California.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Jolly
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D043525:Cytosine Deaminase / Q000235:genetics; D004686:Encephalomyocarditis virus / Q000235:genetics*; D005260:Female; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D000067556:Internal Ribosome Entry Sites / Q000235:genetics*; D008807:Mice, Inbred BALB C; D008979:Moloney murine leukemia virus / Q000235:genetics*; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000235:genetics; D019076:Transgenes / Q000235:genetics*
chemical_list: D000067556:Internal Ribosome Entry Sites; D012367:RNA, Viral; D049452:Green Fluorescent Proteins; D043525:Cytosine Deaminase