PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints.
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 phiC31 integrase to bring about unidirectional genomic integration.


METHODS
Rabbit and human synovial cells were co-transfected with a plasmid expressing phiC31 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 phiC31 integrase were intra-articularly injected into rabbit knees. Joint sections were used for histological analysis of beta-gal expression, and synovial cells were isolated to measure luciferase expression.


RESULTS
We demonstrated that co-transfection of a plasmid expressing phiC31 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 phiC31 integrase.


CONCLUSIONS
The ability of phiC31 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.
Keravala
Annahita
A
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
Portlock
Joylette L
JL
Nash
Joan A
JA
Vitrant
David G
DG
Robbins
Paul D
PD
Calos
Michele P
MP
D000818:Animals; D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D015316:Genetic Therapy; D005822:Genetic Vectors; D015894:Genome, Human; D006801:Humans; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D034982:Integration Host Factors; D007596:Joints / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011817:Rabbits; D013583:Synovial Membrane / Q000166:cytology*; D014162:Transfection; D019076:Transgenes
D005819:Genetic Markers; D034982:Integration Host Factors; D019426:Integrases