Evaluation of Long-Term Gene Expression in Mouse Liver Using PhiC31 Integrase and Hydrodynamic Injection

Abstract

PhiC31 integrase has the potential to achieve long-term transgene expression because of site-specific and unidirectional recombination. In this study, plasmid DNA (pDNA) encoding Gaussia luciferase (Gluc) cDNA was constructed and the optimal condition for long-term gene expression using phiC31 integrase in mouse liver after hydrodynamic injection was investigated. Gluc is secreted and thus allows quantification of its expression level in blood and easy determination of the expression time-course. Mice were co-transfected with 25 µg donor pDNA (pORF-Gluc/attB) and different amounts of helper pDNA (pCMV-int; from 1 to 50 µg). Serum Gluc expression was evaluated during 120 d. The most highly sustained gene expression was obtained when 10 µg of helper pDNA was co-transfected in ICR and C57BL/6 mice. However, the Gluc expression in C57BL/6 mice was slightly lower and less durable than that in ICR mice. Little hepatic damage caused by phiC31 integrase was observed during 120 d in ICR and C57BL/6 mice.Key words non-viral gene delivery; gene vector; plasmid DNA; hepatocyte A variety of strategies for long-term gene expression have been developed. However, many obstacles that hinder safe and effective gene therapy remain. For example, retroviral vectors, which induce highly efficient integration, mediate random integration into genomic DNA that activated oncogenes both in animals and in clinical trials.1-3) Transposons have been used as an effective method of genomic integration in vivo, but they also integrate randomly.4) Therefore, the use of site-specific recombinases such as Cre and Flp could be a promising method; however, their main utility is in creating deletions mediated by the reversibility of their enzyme reactions. 5,6) It was reported that bacteriophage phiC31 integrase performs a site-specific and unidirectional reaction. 7) In nature, phiC31 integrase mediates the recombination of the phage genome into the bacterial chromosome via a reaction between the phage attP site and the bacterial attB site. 8) Recently, Thyagarajan et al. have demonstrated that phiC31 integrase can act in cultured mammalian cells and that it can efficiently recombine plasmid DNA (pDNA) that includes an attB site into so called pseudo attP sites in mammalian genomic DNA.9) In addition, the number of potential sites integrated by phiC31 integrase in the human genome was estimated at ca. 370 sites, and the sites appear to be in comparatively safe locations. 10)So far, the use of phiC31 integrase for prolonged gene expression has been demonstrated in mice [11][12][13] and in cultured cells. 9,14) This system requires the use of two types of pDNAs: the donor pDNA encoding attB and the helper pDNA encoding phiC31 integrase. Hydrodynamic injection is a transfection method that permits high gene expression in the liver and there are many reports combining this method with phiC31 integrase for basic research using firefly luciferase, genes related to genetic diseases or hepatic murine cancer model. 11,[15][16][17][18][19][20][21] Taking thes...

Kidney-selective gene transfection using anionic bubble lipopolyplexes with renal ultrasound irradiation in mice

“…Long-term gene expression in the liver and lungs has been observed using CpG-reduced pDNA, a hepatitis B virus promoter, an albumin promoter, and other transposon and integrase systems. 24,25,[42][43][44][45] To date, however, little is known about the long-term expression of transfected genes in the kidney. We therefore constructed pDNA under the control of a CAG promoter, which has shown strong gene expression in various cell lines, such as COS7 and 293 cells, in murine embryonic stem cells, and even in vivo.…”

Hydrodynamic Delivery: Characteristics, Applications, and Technological Advances

“…Minicircle DNA [33,114,124,132,136,137,143,171] US-targeted microbubble destruction [178] PhiC31 Integrase [197] microRNA [47,150,152,[188][189][190][191] Computer-assisted hydrodynamic delivery [12,198] Sleeping Beauty [30,96,111,142,191,[199][200][201][202] Circular RNA [126] Bioluminescence imaging [47,[203][204][205][206] piggyBac [126,207] shRNA [94,192,193] Tissue clearing [206] Cre-loxP [208][209][210] siRNA [87,[194][195][196] Repopulation [189,211] CreER…”

“…Aiming at longlasting gene expression using nonviral vectors and in addition to epigenetic control [227], plasmids that replicate in an episomal fashion and site-specific integration systems were developed and hydrodynamically delivered. In terms of site-specific integration, PhiC31 integrase [197], sleeping beauty transposon [30,96,111,142,191,[199][200][201][202], piggyBac transposon [126,207], and Cre-loxP and technologies derived from it [208][209][210] were a major focus. Tamoxifen-dependent Cre recombinase (CreER) can initiate the recombination event at any desired time point [201,212].…”

Improved Lentiviral Gene Delivery to Mouse Liver by Hydrodynamic Vector Injection through Tail Vein

“…The injected DNA solution passes through the sinusoidal structure to the portal veins and enters the hepatocytes through transient pores formed in the cell membrane. 3 While hydrodynamic injection of naked DNA offers a simple and safe (non-viral) way to deliver genetic cargo to liver tissue and reaches an efficacy where nearly half of all hepatocytes are being targeted, 4 this method suffers from its transient nature and gene expression drops rapidly, 4 , 5 , 6 although inclusion of control regions may provide prolonged episomal expression. 7 Gene delivery using viral vectors may offer prolonged transgene expression, and both adenovirus-, adeno-associated virus (AAV), and lentivirus-derived vectors have been adapted for potent gene transfer to liver tissue.…”