Hydrodynamics-based Injection Research Articles

Monitoring method for transgene expression in target tissue by blood sampling

In this study, we have developed a novel method to monitor transgene expression in tissues by blood sampling. We administered plasmid DNA (pDNA) encoding non-secretory form of firefly luciferase as a reporter gene and pDNA encoding secretable Gaussia princeps luciferase as a monitor gene simultaneously into mice. Good positive correlations were found between log-transgene expression of the reporter gene and the monitor gene in the treated muscle, between the monitor gene in the treated muscle and plasma, and consequently between the reporter gene in the treated muscle and the monitor gene in plasma after naked pDNA transfer into the muscle of mice. Such positive correlations were also found with gastric serosal surface instillation of naked pDNA, intravenous injection of lipoplex, and hydrodynamics-based injection of naked pDNA. We developed monitoring method of transgene expression in tissues by blood sampling, which was named ‘Therapeutic transgene monitoring (TTM)’, after ‘Therapeutic drug monitoring (TDM)’.

The effect of miR-155 on HBV replication and PTEN expression in vivo

Objective: To construct the mmu-miR-155 eukaryotic overexpression vector pmR-155 and to investigate its effect on HBV replication and expression of PTEN in vivo. Methods: The mmu-mir-146a precursor gene fragment pre-mmu-mir-146a was amplified by PCR, then connected to the pmR-mCherry plasmid vector after double enzyme digestion, the accuracy of recombinant vector was verified by colony PCR、double enzyme digestion and sequencing; then the recombinant vector was transfected HBV transgene mice(Experimental Group)with hydrodynamics-based injection via vena caudalis, and pmR-mCherry plasmid、PBS were respectively transfected into the mice as Empty plasmid Group、Blank Group. The concentration of IFN-γ in the serum was detected by ELISA. The expression of SOCS1、PTEN mRNA in the liver was detected by qPCR at 30d post-transfectioned. The Western blot was performed to detect the changes in SOCS1、PTEN、HBX in the liver tissue at 30 d post-transfectioned. The results were analyzed with Student's t-test, or one-way analysis of variance and the least significant difference test. Results: the colony PCR、double enzyme digestion and sequencing verified that the gene was inserted into the pmR-mCherry vector. Compared with Blank Group, the expression of miR-155 in the Experimental Group was significantly increased(t = 8.90, P < 0.01); the concentration of IFN-γ in the Experimental Group was significantly increased(F = 26.58, P < 0.01); the mRNA(F(SOCS1 mRNA) = 19.72, P < 0.01; F(PTEN mRNA) = 7.38, P < 0.05) and protein(F(SOCS1) = 50.30, P < 0.01; F(PTEN) = 129.00, P < 0.01) expression of COCS1、PTEN was significantly decreased in the Experimental group and the protein of HBX was also significantly(F(HBX) = 77.97, P < 0.01). Conclusion: The pmR-155 eukaryotic overexpression vector is successfully constructed, this recombinant vector can express miR-155 stably; miR-155 can down-regulate cocs1、PTEN gene expression and up-regulate the expression of IFN-γ, it can inhibit the replication of HBV and a potential targets to treating hepatocellular carcinoma.

Protective effects of hydrodynamics-based injection preconditioning on hepatic warm ischemia and reperfusion injury in mice

Objective To investigate the effects of hydrodynamics-based injection preconditioning on hepatic warm ischemia and reperfusion (I/R) injury in mice.Methods Sixty male BALB/c mice were randomly divided into four groups:(1) sham group; (2) warm I/R group; (3) hydrodynamics-based injection (HD) + I/R group:Hydrodynamics-based injection (5-7 s) was performed at 72 h before isehemia with normal saline in a large volume of 2.5 mL; (4) routine injection (RT) + I/R group:Routine tail vein injection (＞30 s) was performed at 72 h before isehemia with normal saline in a large volume of 2.5 mL.The model of hepatic warm L/R was produced by clamping the hepatic portal for 45 min,followed by 6 h reperfusion.After reperfusion,blood and liver samples were collected.The levels of serum alanine aminotransferase (ALT),aspartate aminotransferase (AST) and hepatic malondialdehyde (MDA) were measured,and the histologic changes of the liver tissue were observed.Results The levels of serum ALT,AST and hepatic MDA in the HD + L/R group were (641.60 ±225.00) U/L,(1180.80 ±212.54) U/L,and (11.00 ± 1.06) mmol/L respectively,significantly lower than in RT + I/R group [(1716.20 ±283.50) U/L,(1994.60±303.80) U/L,(17.22 ±1.22) mmol/L] and L/R group [(1661.20±306.38) U/L,(2046.60±719.21) U/L,(17.20±2.09) mmol/L] (P＜0.05).Meanwhile,the histology revealed hepatocyte necrosis and infiltration of inflammatory cells were significantly alleviated in HD + I/R group.Conclusion Hydrodynamics-based injection preconditioning significantly attenuated hepatic warm I/R injury in mice. Key words: Tail vein injection; Hepatic; Warm ischemia; Reperfusion injury; Preconditioning

A designed curved DNA sequence remarkably enhances transgene expression from plasmid DNA in mouse liver

The intranuclear disposition of plasmid DNA is extremely important for transgene expression. The interactions between the plasmid DNA and the histone proteins are one of the keys for controlling the disposition. In this study, the effects of a left-handedly curved sequence (20-40 repeated A•T tracts) on transgene expression from a plasmid were examined in vivo. A naked luciferase plasmid with the curved sequence was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. Interestingly, transgene expression was markedly increased by the addition of the curved sequence. An analysis of the nucleosome positions near the left-handedly curved sequence suggested that the sequence functions as an acceptor of the histone core and allows nucleosome sliding, resulting in transcriptional activation. These results suggested that the designed curved DNA sequences could control transgene expression from plasmid DNAs in vivo.

Liver-targeted siRNA delivery by polyethylenimine (PEI)-pullulan carrier

Recently, small interfering RNA (siRNA)-based therapeutics have been used to treat diseases. Efficient and stable siRNA delivery into disease cells is important in the use of this agent for treatment. In the present study, pullulan was introduced into polyethylenimine (PEI) for liver targeting. PEI/siRNA or pullulan-containing PEI/siRNA complexes were delivered into mice through the tail vein either by a hydrodynamics- or non-hydrodynamics-based injection. The incidence of mortality was found to increase with an increase in the nitrogen/phosphorus (N/P) ratio of PEI/siRNA complexes. Moreover, the hydrodynamics-based injection increased mice mortality. Introduction of pullulan into PEI dramatically reduced mouse death after systemic injection. After systemic injection, the PEI/fluorescein-labeled siRNA complex increased the level of fluorescence in the lung and the PEI-pullulan/siRNA complex led to an increased fluorescence level in the liver. These results suggest that the PEI-pullulan polymer may be a useful, low toxic means for efficient delivery of siRNA into the liver.

Effects of non-B DNA sequences on transgene expression

DNA conformation may be an important factor affecting gene transcription. In this study, we examined how DNA sequences with unusual conformations affect transgene expression. A(30) and (CG)(15) sequences that can adopt the B' and Z conformations, respectively, were introduced into a beta-actin promoter. Luciferase plasmids containing the manipulated promoter were transfected into NIH3T3 cells by electroporation and were delivered into mouse livers with a hydrodynamics-based injection. Expression from plasmid with the (CG)(15) sequence was multiple times higher than expression from control plasmid DNA. The A(30) sequence also tended to enhance expression. These results suggest that non-B DNA sequences could improve transgene expression in cells.

Therapeutic RNA silencing of Cys-X3-Cys chemokine ligand 1 gene prevents mice from adenovirus vector-induced acute liver injury

Gene therapy using adenovirus vectors may induce acute liver injury. Tissue injury induced by an adenovirus is likely associated with elevated expression of the Cys-X3-Cys chemokine ligand 1 (CX(3)CL1)/fractalkine (FKN) protein at the site of inflammation. However, the extent to which the actions of FKN contribute to liver injury remains unclear. We induced acute liver injury in mice by a hydrodynamics-based injection of adenovirus vector, which was confirmed to depend on the presence of natural killer (NK) cells and NK-dependent interferon-gamma (IFN-gamma). When the transferred adenovirus vector was inserted with the FKN gene, the severity of liver injury increased with much more Cys-X3-Cys chemokine receptor 1 (CX(3)CR1)-positive NK cell recruitment into the liver because of exogenous overproduction of FKN protein. Moreover, when production of endogenous FKN protein was silenced by inserting FKN-small interfering RNA into the adenovirus vector or was neutralized by an FKN-specific antibody, the adenovirus-induced acute severe liver injury was notably prevented with much lower hepatic NK cell infiltration and a significant reduction in the serum levels of IFN-gamma. Our findings suggest a strategy to prevent or alleviate adenovirus vector-induced acute liver injury by blocking FKN-CX(3)CR1 interaction in adenovirus vector-based gene therapy.

Transient activation of transgene expression by hydrodynamics-based injection may cause rapid decrease in plasmid DNA expression

The intranuclear disposition of exogenous DNA is quite important for the therapeutic effects of the administered DNA. The expression efficiency from one copy of exogenous DNA delivered by hydrodynamics-based injection dramatically decreases over time, and this 'silencing' occurs without CpG methylation. In this study, naked luciferase-plasmid DNA was delivered into mouse liver by hydrodynamics-based injection, and modifications of the histones bound to the plasmid DNA were analyzed by a chromatin immunoprecipitation (ChIP) analysis. In addition, the effects of a second hydrodynamics-based injection on the expression from the plasmid DNA were examined. The ChIP analysis revealed that the modification status of histone H3 remained constant from 4 h to 4 weeks. Surprisingly, the injection of saline without DNA enhanced the luciferase expression from the preexisting DNA administered 4 and 14 days previously. Our results suggest that histone modification plays no role in the silencing. Instead, our data suggest that the transgene expression is activated by the hydrodynamics-based injection manipulation, and that the return from the activated status causes the silencing.

The location of the left-handedly curved DNA sequence affects exogenous DNA expression in vivo

The intranuclear disposition of a plasmid is extremely important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. The location of the left-handedly curved sequence determined the transgene expression, without affecting the amount of intranuclear exogenous DNA. The plasmid containing the curved sequence at the location that results in the exposure of the TATA box out of the nucleosome core showed the highest expression. These results suggest that sequences with high histone affinity could control transgene expression from plasmids in vivo.

Intranuclear disposition of exogenous DNA in vivo: Silencing, methylation and fragmentation

The intranuclear disposition of exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was delivered into mouse liver by a hydrodynamics-based injection, and the amounts of intranuclear plasmid DNA, luciferase, and its mRNA were quantitated at various time points. Methylation of the promoter of the luciferase gene was also analyzed. Expression efficiency from one copy of the exogenous DNA dramatically decreased over time, and the DNA was methylated and degraded into fragments. Unexpectedly, methylation of the intact plasmid DNA was low and did not increase over time. Rather, the fragmented DNA was methylated more frequently than the intact plasmid. These results suggest that the CpG methylation and the degradation of exogenous DNA, and its ‘silencing’, occurred in parallel in the nucleus.

208. Sleeping Beauty Transposon-Mediated Long-Term α-L-iduronidase Expression and Correction of Lysosomal Pathology in the Murine Model of Mucopolysaccharidosis (MPS) Type I

Top of pageAbstract The purpose of this project was to evaluate the Sleeping Beauty (SB) transposon for delivery of the human |[alpha]|-L-iduronidase ( IDUA) gene to chromosomes in the livers of IDUA-deficient mice for long- term expression and correction of MPS type I. We chose the hydrodynamics-based DNA injection, which targets mainly the liver. We constructed SB transposon, plasmid-based vectors and injected them into idua-/-mice. Because our goal was in part to determine the efficacy of transposition as a way of providing long-term expression of IDUA protein, control groups of MPS I mice did not get the transposase. Blood samples for plasma isolation were collected 1 day after treatment and once every 2 weeks thereafter. Plasma IDUA activities reached >100-fold of wild type levels on day 1 following treatment, but were essentially gone in all mice 4 weeks following gene delivery. IDUA activity was not detected in the livers of these mice 3 months after plasmid administration. We examined the duration of the transposon-delivered human IDUA DNA maintenance and enzyme activity over 6 months in the liver of wild type (WT) C57Bl/6 mice that express IDUA. As in the MPS I mice, plasma and liver IDUA activity reached supra-normal levels on day 1 and remained at this level for the first week, but still dropped dramatically within 2 weeks and by 4 weeks were indistinguishable from background. Using DNA PCR, we found that the maintenance of the IDUA transgene mirrored the IDUA activity time-line. Even though transposition was confirmed by an excision assay, the PCR product was detectable only for the first two weeks, but not thereafter. It appears that cells that express the IDUA gene are cleared from the liver of treated mice by 4 weeks following injection, which suggests induction of an immune response either to the therapeutic protein or/and the cells that express the therapeutic gene. This hypothesis is supported by our observations that in all cyclophosphamide immune-suppressed MPS I mice, the initial 1-day plasma activity levels dropped more than 100-fold by 2 weeks, but then persisted at 20-500% WT activity in some mice. IDUA levels were stable (up to 5-fold higher than in the WT mice) in SB-transposase-treated MPS I mice whereas without SB, IDUA levels declined to near-background over 3 months. These persistent IDUA activity levels were sufficient to reduce the number and size of pathologic inclusions in the liver seen by toluidine blue staining. Secondary elevation of murine |[beta]|-glucuronidase, another lysosomal enzyme, was reduced in the liver, spleen and kidney and correlated with degree of reduction of lysosomal pathology in these organs. Thus, with immune suppression, a single dose of the SB transposon system can result in partial to complete biochemical correction of IDUA activity in the livers of treated adult MPS I mice.

In Vivo Functions of Hepatitis C Virus Core Protein: A Study in a Mouse Model Generated by Hydrodynamics-based DNA Injection of the Core-expressing DNA

Hydrodynamics-based DNA injection method was employed to establish a mouse model expressing the hepatitis C virus (HCV) core or the luciferase protein in the liver. The model was used to investigate the in vivo function of HCV core. Our results showed that both HCV core and luciferase proteins were expressed transiently in mouse liver, lasting for about 7～21 days. In the animals injected with core DNA, significant levels of CD4+ and CD8+ T-cells were recruited to the liver and core-specific cytotoxic T-lymphocyte responses were detected in their spleens. The same DNA injection in immunodeficient non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, however, led to prolonged core gene expression, lasting for up to 56 days. Mice expressing the core protein were shown to be more resistant to Fas-mediated death than those without core protein. Our mouse experiments thus indicated that expression of HCV core in mouse liver does not exhibit immunosuppressive effects that can prevent the hepatocytes from immune clearance, but could protect them against Fas-mediated apoptotic death.

566. Silencing of Plasmid DNA after Hydrodynamics-Based Transfection in Mice

Top of pageAbstract Non-viral vectors are promising tools for gene therapy because of their safety profile and are expected to be alternative to viral vectors. However, gene expression efficiency of non-viral vectors is lower than that of viral vectors especially in vivo. This low expression of non-viral vectors is mainly thought to be due to insufficient transgene delivery into the nucleus of target cells, and many efforts have been made to improve the gene delivery. Although establishment of such delivery strategy is one of the most important issues for application of non-viral vectors, delivery of transgenes into the nucleus is not an endpoint of gene therapy. The final goal of non-viral gene therapy is to achieve the persistent and properly regulated expression of transgenes in the target nucleus without disrupting host's nuclear homeostasis. However, it has not been precisely revealed how tansgenes, especially transgenes on episomes, behave in the nucleus. In order to elucidate the fate of plasmid DNA (pDNA) after delivery into the nucleus in vivo, we investigated the time course of gene expression and the copy number of intranuclear pDNA after pDNA transfer to the liver. Here we report a gene silencing phenomenon of a plasmid DNA after delivery into the nucleus. In this study, a pDNA construct encoding luciferase driven by the cytomegalovirus (CMV) promoter was transfected into BALB/c mice using hydrodynamics-based injection, and luciferase expression was monitored for a week. The copy number of intranuclear pDNA was measured by quantitative real-time PCR. In agreement with some previous reports, luciferase expression peaked at 8 hours post injection, and then rapidly reduced (half-life was approximately 10 hours). On the other hand, the nuclear pDNA was gradually eliminated with a half-life of approximately 42 hours, about 4 times as long as that of luciferase expression. Next, the ratio of luciferase activity to the copy number of nuclear pDNA was calculated to directly compare the expression ability of one pDNA molecule at various time points. This ratio peaked at 24 hours post injection, and remarkably, decreased more than 100-fold within a few days. These results suggest that the pDNA was qualitatively changed and rapidly inactivated after delivery into the nucleus. Currently, we are examining the mechanism of such pDNA silencing.