title: Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors.
abstract: Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF) expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF). Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration.
authors.affiliation: Department of Biomedicine, Aarhus University , Aarhus, Denmark.
authors.forename: Anne Louise
authors.initials: AL
authors.lastname: Askou
authors.affiliation: Department of Biomedicine, Aarhus University , Aarhus, Denmark.
authors.forename: Lars
authors.initials: L
authors.lastname: Aagaard
authors.affiliation: Department of Ophthalmology, Unit of Gene Therapy and Stem Cell Biology, University of Lausanne, Jules-Gonin Eye Hospital , Lausanne, Switzerland.
authors.forename: Corinne
authors.initials: C
authors.lastname: Kostic
authors.affiliation: Department of Ophthalmology, Unit of Gene Therapy and Stem Cell Biology, University of Lausanne, Jules-Gonin Eye Hospital , Lausanne, Switzerland.
authors.forename: Yvan
authors.initials: Y
authors.lastname: Arsenijevic
authors.affiliation: Department of Biomedicine, Aarhus University , Aarhus, Denmark.
authors.forename: Anne Kruse
authors.initials: AK
authors.lastname: Hollensen
authors.affiliation: Department of Ophthalmology, Aarhus University Hospital , Aarhus, Denmark.
authors.forename: Toke
authors.initials: T
authors.lastname: Bek
authors.affiliation: Department of Biomedicine, Aarhus University , Aarhus, Denmark.
authors.forename: Thomas Gryesten
authors.initials: TG
authors.lastname: Jensen
authors.affiliation: Department of Biomedicine, Aarhus University , Aarhus, Denmark.
authors.forename: Jacob Giehm
authors.initials: JG
authors.lastname: Mikkelsen
authors.affiliation: Department of Biomedicine, Aarhus University , Aarhus, Denmark.
authors.forename: Thomas Juhl
authors.initials: TJ
authors.lastname: Corydon