Assuntos
Ensaio de Imunoadsorção Enzimática , Eritropoetina/análise , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Reações Cruzadas , Eritropoetina/sangue , Eritropoetina/genética , Eritropoetina/imunologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Imunoglobulina G/imunologia , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Kit de Reagentes para Diagnóstico , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/imunologia , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
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.
Assuntos
Cromossomos Humanos Par 19 , DNA Helicases/genética , Proteínas de Ligação a DNA , Dependovirus/genética , Transativadores/genética , Integração Viral , Linhagem Celular , Núcleo Celular/metabolismo , DNA Helicases/metabolismo , Replicação do DNA/genética , Vetores Genéticos , Células HeLa , Humanos , Ligantes , Mifepristona/farmacologia , Receptores de Progesterona/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/metabolismoRESUMO
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.
