Keravala Annahita, Portlock Joylette L, Nash Joan A, Vitrant David G, Robbins Paul D, Calos Michele P
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
J Gene Med. 2006 Aug;8(8):1008-17. doi: 10.1002/jgm.928.
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.
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.
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.
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.
基因转移至关节滑膜已被证明是治疗类风湿关节炎（RA）及相关关节疾病的有效方法。然而，基因递送的效率和持续时间一直限制着关节炎基因治疗的成功。非病毒基因递送常伴随的瞬时基因表达可通过将载体DNA整合到宿主基因组中得以延长。我们报告了一种用于关节非病毒基因治疗的新方法，该方法利用噬菌体phiC31整合酶实现单向基因组整合。
将表达phiC31整合酶的质粒与含有转基因和attB位点的质粒共转染兔和人滑膜细胞。在有或无G418选择的情况下培养细胞，分别测定新霉素抗性菌落数或eGFP细胞数。进行质粒拯救、PCR查询和DNA序列分析以揭示兔和人基因组中的整合位点。对于体内研究，将attB报告基因质粒和表达phiC31整合酶的质粒关节腔内注射到兔膝关节。关节切片用于β-半乳糖苷酶表达的组织学分析，分离滑膜细胞以测量荧光素酶表达。
我们证明，将表达phiC31整合酶的质粒与含有转基因和attB的质粒共转染可增加兔滑膜成纤维细胞和原代人RA滑膜细胞中转基因表达的频率。质粒-染色体连接处的质粒拯救和DNA序列分析揭示了在兔基因组内源性假attP序列处的整合，PCR查询检测到在人基因组中先前表征的整合位点处的整合。关节腔内注射attB报告基因质粒和表达phiC31整合酶的质粒后，在兔膝关节体内检测到显著更高水平的转基因表达。
phiC31整合酶促进滑膜细胞基因组整合并增加兔滑膜中转基因表达的能力表明，与更有效的DNA递送方法相结合，该整合酶系统可能有益于类风湿关节炎和其他关节疾病的治疗。
