title: Transduction and utility of the granulocyte-macrophage colony-stimulating factor gene into monocytes and dendritic cells by adeno-associated virus.
abstract: The genetic manipulation of antigen-presenting dendritic cells (DC) offers promise for stimulating the immune response, in particular for anticancer and antiviral protocols. As adeno-associated virus (AAV) has shown promise as a gene delivery vector for transducing a variety of hematopoietic cell types, we have investigated AAV's ability to genetically alter DC. In this analysis, we modified the standard granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) treatment of adherent monocytes to generate DC. In our protocol, adherent monocytes were first infected with an AAV/GM-CSF/Neo vector, and the addition of IL-4 was delayed for 2 days to allow for a brief period of monocyte proliferation. AAV-mediated transduction of the GM-CSF and Neo genes into monocytes/DC precursors was demonstrated by G418 selection, GM-CSF secretion, GM-CSF RNA expression (reverse transcriptase-polymerase chain reaction amplification [RT-PCR]), and cell proliferation. Cells resulting from infection with AAV/GM-CSF/Neo virus, and subsequent IL-4 and tumor necrosis factor-alpha (TNF-alpha) treatment, displayed multiple classic markers consistent with mature DC. Finally, chromosomal integration of the AAV vector was also demonstrated in sorted CD83+ DC. These data strongly suggest that AAV vectors will be useful for the genetic manipulation of DC and suggest that the transduction of the GM-CSF gene was able to fully replace the need for exogenous GM-CSF in the production of mature DC.
authors.affiliation: Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: A D
authors.initials: AD
authors.lastname: Santin
authors.forename: M
authors.initials: M
authors.lastname: Mane
authors.forename: M
authors.initials: M
authors.lastname: Chiriva-Internati
authors.forename: G P
authors.initials: GP
authors.lastname: Parham
authors.forename: A
authors.initials: A
authors.lastname: Ravaggi
authors.forename: P L
authors.initials: PL
authors.lastname: Hermonat
mesh_terms: D017951:Antigen Presentation; D015703:Antigens, CD; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics* / Q000502:physiology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007136:Immunoglobulins / Q000032:analysis; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000032:analysis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D016662:Virus Integration; D000097802:CD83 Antigen
chemical_list: D015703:Antigens, CD; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D000097802:CD83 Antigen