Subject(s)
Enzyme-Linked Immunosorbent Assay , Erythropoietin/analysis , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , Cross Reactions , Erythropoietin/blood , Erythropoietin/genetics , Erythropoietin/immunology , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Immunoglobulin G/immunology , Injections, Intramuscular , Mice , Mice, Inbred BALB C , Reagent Kits, Diagnostic , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/immunology , Sensitivity and Specificity , Species SpecificityABSTRACT
It is of great interest for gene therapy to develop vectors that drive the insertion of a therapeutic gene into a chosen specific site on the cellular genome. Adeno-associated virus (AAV) is unique among mammalian viruses in that it integrates into a distinct region of human chromosome 19 (integration site AAVS1). The inverted terminal repeats (ITRs) flanking the AAV genome and the AAV-encoded nonstructural proteins Rep78 and/or Rep68 are the only viral elements necessary and sufficient for site-specific integration. However, it is also known that unrestrained Rep activity may cause nonspecific genomic rearrangements at AAVS1 and/or have detrimental effects on cell physiology. In this paper we describe the generation of a ligand-dependent form of Rep, obtained by fusing a C-terminally deleted Rep68 with a truncated form of the hormone binding domain of the human progesterone receptor, which does not bind progesterone but binds only its synthetic antagonist RU486. The activity of this chimeric protein, named Rep1-491/P, is highly dependent on RU486 in various assays: in particular, it triggers site-specific integration at AAVS1 of an ITR-flanked cassette in a ligand-dependent manner, as efficiently as wild-type Rep68 but without generating unwanted genomic rearrangement at AAVS1.
Subject(s)
Chromosomes, Human, Pair 19 , DNA Helicases/genetics , DNA-Binding Proteins , Dependovirus/genetics , Trans-Activators/genetics , Virus Integration , Cell Line , Cell Nucleus/metabolism , DNA Helicases/metabolism , DNA Replication/genetics , Genetic Vectors , HeLa Cells , Humans , Ligands , Mifepristone/pharmacology , Receptors, Progesterone/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Trans-Activators/metabolismABSTRACT
Adeno-associated virus (AAV) integrates very efficiently into a specific site (AAVS1) of human chromosome 19. Two elements of the AAV genome are sufficient: the inverted terminal repeats (ITRs) and the Rep78 or Rep68 protein. The incorporation of the AAV integration machinery in nonviral delivery systems is of great interest for gene therapy. We demonstrate that purified recombinant Rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome Lipofectamine, promoting the rescue-replication of a codelivered ITR-flanked cassette in adenovirus-infected cells and its site-specific integration in noninfected cells. The sequencing of cloned virus-host DNA junctions confirmed that lipofected Rep68 protein triggers site-specific integration at the same sites in chromosome 19 already characterized in cells latently infected with AAV.
