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PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints
Author(s) -
Keravala Annahita,
Portlock Joylette L.,
Nash Joan A.,
Vitrant David G.,
Robbins Paul D.,
Calos Michele P.
Publication year - 2006
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.928
Subject(s) - plasmid , integrase , biology , microbiology and biotechnology , transfection , transgene , gene , reporter gene , genetic enhancement , synovial membrane , gene expression , genetics , arthritis , immunology
Background Gene transfer to synovium in joints has been shown to be an effective approach for treating pathologies associated with rheumatoid arthritis (RA) and related joint disorders. However, the efficiency and duration of gene delivery has been limiting for successful gene therapy for arthritis. The transient gene expression that often accompanies non‐viral gene delivery can be prolonged by integration of vector DNA into the host genome. We report a novel approach for non‐viral gene therapy to joints that utilizes phage ϕC31 integrase to bring about unidirectional genomic integration. Methods Rabbit and human synovial cells were co‐transfected with a plasmid expressing ϕC31 integrase and a plasmid containing the transgene and an attB site. Cells were cultured with or without G418 selection and the number of neo‐resistant colonies or eGFP cells determined, respectively. Plasmid rescue, PCR query, and DNA sequence analysis were performed to reveal integration sites in the rabbit and human genomes. For in vivo studies, attB ‐reporter gene plasmids and a plasmid expressing ϕC31 integrase were intra‐articularly injected into rabbit knees. Joint sections were used for histological analysis of β‐gal expression, and synovial cells were isolated to measure luciferase expression. Results We demonstrated that co‐transfection of a plasmid expressing ϕC31 integrase with a plasmid containing the transgene and attB increased the frequency of transgene expression in rabbit synovial fibroblasts and primary human RA synoviocytes. Plasmid rescue and DNA sequence analysis of plasmid‐chromosome junctions revealed integration at endogenous pseudo attP sequences in the rabbit genome, and PCR query detected integration at previously characterized integration sites in the human genome. Significantly higher levels of transgene expression were detected in vivo in rabbit knees after intra‐articular injection of attB ‐reporter gene plasmids and a plasmid expressing ϕC31 integrase. Conclusion The ability of ϕC31 integrase to facilitate genomic integration in synovial cells and increase transgene expression in the rabbit synovium suggests that, in combination with more efficient DNA delivery methods, this integrase system could be beneficial for treatment of rheumatoid arthritis and other joint disorders. Copyright © 2006 John Wiley & Sons, Ltd.