Long-term expression of human alpha1-antitrypsin gene in mouse liver achieved by intravenous administration of plasmid DNA using a hydrodynamics-based procedure

The liver is an important target organ for gene transfer due to its large capacity for synthesizing serum proteins and its involvement in numerous genetic and acquired diseases. Previously, we and others have shown that an efficient gene transfer to liver cells in vivo can be achieved by an intravenous injection of plasmid DNA using a hydrodynamics-based procedure. In this study, we systematically characterized the expression of transgene encoding a secretory protein in mouse. Using human alpha1-antitrypsin (hAAT) gene as a reporter, we demonstrate that the serum level of hAAT can reach as high as 0.5 mg/ml by a simple tail vein injection of 10-50 microg plasmid DNA into a mouse. The serum hAAT reaches the peak level 1 day after DNA injection and then declines during the following 2 to 4 weeks to 2-5 microg/ml, a level which persists for at least 6 months. Southern analysis of extracted DNA and RT-PCR analysis of RNA from the liver reveal that hAAT gene is active and present as episomal form after 6 months. These results suggest that the hydrodynamics-based transfection procedure provides a valuable tool for screening genes for therapeutic purposes in whole animals.     

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.     

Electric gene transfer to the liver following systemic administration of plasmid DNA

Recently, there has been an increasing level of interest in electroporation for gene delivery due to the site-specific nature of the delivery, as well as the high efficiency of the method. Electroporation involves the application of a pulsed electric field to cells to enhance cell permeability, resulting in the transit of exogenous polynucleotide across the cytoplasmic membrane. Electroporation is traditionally performed by locally injecting DNA to the site of interest followed by the application of electric field. Compared with the local injection of plasmid DNA to the liver, systemic injection has the advantage of delivering genes to more hepatocytes. We describe here a method for efficient gene transfer to the liver by electroporation following tail vein administration of the naked DNA. The cells expressing the reporter gene are more broadly distributed with this systemic injection, as compared with direct injection.     

Hepatocyte-targeted in vivo gene expression by intravenous injection of plasmid DNA complexed with synthetic multi-functional gene delivery system

To achieve hepatocyte-targeted in vivo gene expression, a carrier that controls both the tissue and intracellular distribution of DNA was designed and synthesized. A cationic polymer, poly(L-ornithine) (pOrn), was modified first with galactose, then with a fusigenic peptide (mHA2) to obtain Gal-pOrn-mHA2. When applied with Gal-pOrn-mHA2 to asialoglycoprotein receptor-positive cells, fluorescein-labeled DNA showed a diffuse profile, suggesting the release of DNA from endosomes and/or lysosomes by the carrier. Then the biodistribution and gene expression after intravenous injection of DNA complexes (10 microg DNA per mouse) were examined. After injection of [32P]DNA/Gal-pOrn-mHA2, about 60% of the radioactivity was recovered in the liver, mostly in parenchymal cells. A large amount (81 ng/g tissue) of transgene product (luciferase) was detected in the liver of mice injected with DNA/Gal-pOm-mHA2, which was 280-fold greater than that obtained with DNA/DOTMA:Chol liposomes (50 microg DNA). Prior administration of galactosylated albumin reduced the gene expression to 1/100, indicating the asialoglycoprotein receptor-mediated gene transfer in liver parenchymal cells, ie hepatocytes. The luciferase activity in hepatocytes contributed more than 95% of the total activity in all the tissues examined. Thus, hepatocyte-targeted in vivo gene expression was achieved by the intravenous injection of DNA complex with the multifunctional gene carrier.     

Ultrasound-mediated transfection of canine myocardium by intravenous administration of cationic microbubble-linked plasmid DNA.

We tested the hypothesis that targeted disruption of cationic microbubble-linked plasmid DNA, using diagnostic ultrasound, may aid transfection of large animal myocardium. Plasmid DNA encoding for CAT (pCAT, chloramphenicol acetyltransferase) was bound to a novel cationic microbubble containing MRX-225 for intravenous administration, and 16 dogs in 4 groups variously received this conjugate or plasmid only, or were exposed to ultrasound. Histochemical staining and enzyme-linked immunosorbent assay analysis showed CAT activity in the myocardium of only those animals that received microbubble-linked DNA and were exposed to ultrasound. Thus, disruption of cationic-linked, low-dose plasmid systems by diagnostic ultrasound may facilitate transfection of large animal hearts.     

鼠尾静脉流体力学转染技术对绿色荧光蛋白表达质粒器官靶向分布的影响

目的:观察流体力学尾静脉注射对绿色荧光蛋白基因器官靶向性的影响,为今后质粒载体的基因治疗和功能研究寻找潜在的靶器官。方法:实验于2005-12/2006-04在江西省分子医学重点实验室完成。选用健康雄性昆明鼠40只,将32只小鼠按随机数字表法分为流体力学注射和常规注射两大组,每大组再分为转染组和对照组两个小组(n=8),并设正常对照组(n=8)。①流体力学转染组将100μg/只绿色荧光蛋白表达质粒溶液2mL在5s内快速注入尾静脉;对照组仅在5s内注入林格氏液2mL。②常规注射组则将2mL林格氏液或绿色荧光蛋白表达质粒溶液在30s左右注入尾静脉。注射结束后24h采集各组小鼠血清检测转氨酶,并采集肝、脾、心、肾、肺和脑组织进行冰冻切片,部分肝组织采用多聚甲醛固定后切片,荧光显微镜下观察。结果:40只小鼠全部进入结果分析,无脱失。①流体力学注射组和常规注射组小鼠血清转氨酶与正常对照组比较差异均无显著性意义(P>0.05)。②常规尾静脉注射引起少数肾小球细胞表达绿色荧光蛋白,而肝、脾、心、肺及脑等组织未见明显绿色荧光蛋白表达。③流体力学注射引起肝内绿色荧光蛋白高水平表达,肝细胞表达率接近45%,其他组织则无绿色荧光蛋白表达。结论:流体力学方法是肝靶向性的活体基因转染方法,绿色荧光蛋白可作为该方法进行目的基因研究的一个可靠和方便的示踪剂。     

Polyethylenimine-mediated cellular uptake,nucleus trafficking and expression of cytokine plasmid DNA

Although polyethylenimine (PEI) has been widely used as a nonviral vector, there is little mechanistic understanding on PEI-mediated delivery. Here, we studied whether the expression of murine interleukin-2 (mIL-2) plasmids could be improved by complexation with PEI at various N/P ratios, and whether the cellular uptake, nuclear translocation, and retention of plasmids could be affected by the N/P ratios. Compared with the naked mIL-2, PEI/mIL-2 complexes showed at least two orders of magnitude higher expression at Raw264 cells in the N/P ratio-dependent manner. PEI-mediated cellular uptake and nuclear trafficking of plasmids, quantitated by competitive polymerase chain reaction, also depended on the N/P ratios showing the highest cell and nuclear levels of plasmids at 10/1. The higher cellular levels of plasmid DNA after PEI-mediated delivery were also observed in other cell lines. Unlike naked plasmids, PEI/mIL-2 complexes (N/P ratios >/=4/1) showed prolonged cellular and nuclear retention of mIL-2 plasmids. The nuclear translocation and higher cellular level of plasmids given in PEI complexes were similarly observed by fluorescence microscopy. Moreover, PEI/mIL-2 complexes revealed high stability against DNase I, partly explaining the prolonged subcellular retention. These results indicate that the expression of plasmid mIL-2 might be highly enhanced by complexation with PEI and that such increased expression could be attributed by the higher cellular uptake, nuclear translocation and prolonged retention.     

Safe and effective regulation of hematocrit by gene gun administration of an erythropoietin-encoding DNA plasmid

This work examines the effect of delivering a DNA plasmid encoding murine erythropoietin (pVRmEpo) to BALB/c mice by gene gun. Whereas intramuscular injection elicits a rise in hematocrit persisting >8 months, intradermal delivery triggers the dose-dependent secretion of biologically active erythropoietin (Epo) for approximately 1 month. Repeated administration of pVRmEpo by gene gun elicits a stable increase in hematocrit. The source of the Epo produced following gene gun delivery was analyzed by periodically grafting the site of injection onto naive recipients. Results indicate that both stationary cells (presumably keratinocytes) and migratory (presumably dendritic) cells were transfected and secreted biologically active Epo in vivo. Gene gun administration of plasmid DNA appears to be safe, and provides an additional strategy for achieving the regulated secretion of an exogenous gene product.     

Detection of plasmid DNA vectors following gene transfer to the murine airways

Non-viral gene therapy is being considered as a treatment for cystic fibrosis. In clinical studies and in studies using the mouse airways as a model, current formulations result in only transient transgene expression. A number of reasons for this have been proposed including the loss of plasmid DNA from cells. The aim of these studies was to investigate why transgene expression from non-viral vectors is transient in the mouse lung. Plasmid DNA encoding the luciferase reporter gene was complexed with the cationic lipid GL67 and delivered to the mouse airways. The persistence of plasmid DNA in the mouse lungs was investigated using quantitative PCR and Southern hybridization. Results showed that intact plasmid DNA persisted in the mouse lung in the absence of any detectable luciferase activity. The de novo methylation of plasmid DNA in vivo was investigated as a potential cause of this transient gene expression but results suggested that plasmid DNA does not become de novo methylated in the mouse lung. Therefore processes other than the loss of plasmid DNA from the lung or the de novo methylation of plasmid DNA vectors must be responsible for the transient transgene expression.     

Hepatic and renal dysfunction following nafcillin administration.

OBJECTIVE: To review four cases of combined hepatic and renal toxicity that may be associated with the administration of nafcillin in adults. This type of adverse event with the use of nafcillin has not been previously documented in the literature. DATA SOURCES: References from pertinent articles are identified throughout the text. DATA SYNTHESIS: Nafcillin is a widely used penicillinase-resistant penicillin. In four patients receiving nafcillin doses greater than 9 g/24 hours, changes in renal and hepatic function markers were noted within 72 hours of the initiation of nafcillin therapy. Laboratory values returned toward baseline when nafcillin therapy was discontinued. Elevations in blood urea nitrogen, creatinine, total bilirubin, and lactate dehydrogenase have been previously described in the literature for penicillin-like agents other than nafcillin. The exact mechanism for such toxicities as well as patient risk factors have not been clearly established. CONCLUSIONS: Caution should be taken when initiating nafcillin therapy. Evaluation of renal and liver function tests prior to initiating nafcillin therapy and within the first 72 hours appears warranted. If hepatic and/or renal toxicity is observed, discontinuation of nafcillin should be considered.     

Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.

Development of methods that allow an efficient expression of exogenous genes in animals would provide tools for gene function studies, treatment of diseases and for obtaining gene products. Therefore, we have developed a hydrodynamics-based procedure for expressing transgenes in mice by systemic administration of plasmid DNA. Using cDNA of luciferase and beta-galactosidase as a reporter gene, we demonstrated that an efficient gene transfer and expression can be achieved by a rapid injection of a large volume of DNA solution into animals via the tail vein. Among the organs expressing the transgene, the liver showed the highest level of gene expression. As high as 45 microg of luciferase protein per gram of liver can be achi- eved by a single tail vein injection of 5 microg of plasmid DNA into a mouse. Histochemical analysis using beta-galactosidase gene as a reporter reveals that approximately 40percent of hepatocytes express the transgene. The time-response curve shows that the level of transgene expression in the liver reaches the peak level in approximately 8 h after injection and decreases thereafter. The peak level of gene expression can be regained by repeated injection of plasmid DNA. These results suggest that a simple, convenient and efficient method has been developed and which can be used as an effective means for studying gene function, gene regulation and molecular pathophysiology through gene transfer, as well as for expressing proteins in animals.     

Aminoglycoside uptake increased by tet gene expression

The expression of extrachromosomal tet genes not only confers tetracycline resistance but also increases the susceptibilities of gram-negative bacteria to commonly used aminoglycoside antibiotics. We investigated the possibility that tet expression increases aminoglycoside susceptibility by increasing bacterial uptake of aminoglycoside. Studies of [3H]gentamicin uptake in paired sets of Escherichia coli HB101 and Salmonella typhimurium LT2 expressing and not expressing tet showed that tet expression accelerates energy-dependent [3H]gentamicin uptake. Increased [3H]gentamicin uptake was accompanied by decreased bacterial protein synthesis and bacterial growth. Increased aminoglycoside uptake occurred whether tet expression was constitutive or induced, whether the tet gene was class B or C, and whether the tet gene was plasmid borne or integrated into the bacterial chromosome. tet expression produced no measurable change in membrane potential, suggesting that tet expression increases aminoglycoside uptake either by increasing the availability of specific carriers or by lowering the minimum membrane potential that is necessary for uptake.     

Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein-Barr virus (EBV)-based plasmid vectors

Naked plasmid DNA (pDNA) injection could become an alternative procedure to viral and nonviral gene delivery systems. We have previously shown that Epstein-Barr virus (EBV)-based plasmid vectors containing the EBV nuclear antigen 1 (EBNA1) gene and the oriP sequence enable quite high and long-lasting expression in various in vitro and in vivo transfection systems. The EBV-based plasmids were intravenously injected into mice via their tail vein under high pressure. A large amount of the marker gene product was expressed in the liver; as much as 320 microg of luciferase was demonstrated per gram of liver at 8 to 24 h after a single injection with 10 microg of DNA. More than 70% of liver cells stained with X-gal when beta-gal gene was transferred. The expression level was significantly higher than that obtained by conventional pDNA lacking the EBNA1 gene and oriP. On day 35 after the transfection, the expression from the EBV-based plasmid was approximately 100-fold stronger than the conventional pDNA gene expression. Both the EBNA1 gene and oriP are a prerequisite for the augmentation of the transfection efficiency. These results suggest that the intravascular transfection with naked EBV-based plasmid may provide a quite efficient, simple and convenient means to transduce therapeutic genes in vivo into the liver.     

A combination of poloxamers increases gene expression of plasmid DNA in skeletal muscle

Intramuscular administration of plasmid DNA is a promising strategy to express therapeutic genes, however, it is limited by a relatively low level of gene expression. We report here that a non-ionic carrier, SP1017, composed of two amphiphilic block copolymers, pluronics L61 and F127, also known as poloxamers, significantly increases intramuscular expression of plasmid DNA. Two reporter genes, luciferase and beta-galactosidase, and one therapeutic gene, erythropoietin, were injected intramuscularly with and without SP1017 into C57Bl/6 and Balb/C mice and Sprague-Dawley rats. SP1017 increased gene expression by about 10-fold and maintained higher gene expression compared with naked DNA. Comparison of SP1017 with polyvinyl pyrrolidone (PVP) showed that SP1017 exhibited a significantly higher efficacy and its optimal dose was 500-fold lower. Experiments with beta-galactosidase using X-gal staining suggested that SP1017 considerably increased plasmid DNA diffusion through the tissue. SP1017 also improved expression of the erythropoietin gene leading to an increase in its systemic level and hematocrits. Previous toxicity studies have suggested that SP1017 has over a 1000-fold safety margin. Poloxamers used in SP1017 are listed in the US Pharmacopeia as inactive excipients and are widely used in a variety of clinical applications. We believe that the described system constitutes a simple and efficient gene transfer method to achieve local or systemic production of therapeutic proteins.     

In vivo release and gene expression of plasmid DNA by hydrogels of gelatin with different cationization extents.

The objective of this paper is to investigate the in vivo release and gene expression of lacZ plasmid DNA (pSV-lacZ) by the hydrogels of cationized gelatin. Gelatin with different cationization extents was prepared by changing the amount of ethylenediamine added to aminize the carboxyl groups of gelatin with a water-soluble carbodiimide. The cationized gelatin prepared was crosslinked by various concentrations of glutaraldehyde (GA) to obtain cationized gelatin hydrogels with different cationization extents as the release carrier of plasmid DNA. When the cationized gelatin hydrogels incorporating 125I-labeled pSV-lacZ were implanted into the femoral muscle of mice, the radioactivity remaining decreased with time and the retention period was prolonged with an increase in the concentration of GA used for hydrogel preparation. In vivo experiments with 125I-labeled cationized gelatin hydrogels revealed that the higher the GA concentration, the longer the in vivo retention period of radioactivity remaining for every cationized gelatin hydrogel. Only for the hydrogels prepared from gelatin with the aminized percentages of 29.7, 41.6, and 47.8 mol.%, the time profile of pSV-lacZ retention correlated well with that of hydrogel retention. The gene expression by the cationized gelatin hydrogels incorporating pSV-lacZ depended on the aminized percentage of gelatin and was significant at the percentage of 41.6 mol.% or higher. It is possible that the pSV-lacZ was complexed with the degraded fragments of cationized gelatin and released with a positive charge, resulting in enhanced gene expression. We conclude that gelatin with a cationization extent of at least 41.6 mol.% is needed for the enhanced in vivo gene expression of plasmid DNA by the hydrogel release system.     

Long-term transgene expression from plasmid DNA gene therapy vectors is negatively affected by CpG dinucleotides.

CpG-reduced, CMV-based plasmid DNA constructs encoding human alpha-galactosidase A and factor IX were injected into C57Bl/6, BALB/c, and CD1 mice using hydrodynamics-based delivery of plasmid DNA (pDNA), and gene expression was monitored for 6 months. Linearized and supercoiled pDNAs were compared for their abilities to support long-term expression and to generate immune responses to the transgene product. In all mouse strains supercoiled CpG-reduced pDNA encoding alpha-galactosidase A and factor IX generated higher and more sustained levels of circulating gene product than their supercoiled CpG-replete analogs. Linearizing supercoiled CpG-reduced pDNA did not significantly increase levels of circulating gene product beyond levels supercoiled CpG-reduced pDNA could achieve. Linearizing supercoiled CpG-replete pDNA vectors significantly increased expression compared to their supercoiled CpG-replete analogs, but the increase was short-lived or subtherapeutic. Regardless of vector, liver depot expression did not elicit significant antibody responses to human alpha-galactosidase A or factor IX. Taken together, these data suggest that a clinically acceptable hydrodynamics-based approach targeting the liver combined with CpG-reduced pDNA vectors may represent a viable option for individuals with hemophilia, a lysosomal storage disease, or other disease in which prolonged depot expression of a therapeutic protein from the liver is desirable.     

Dystonia following administration of intravenous radiographic contrast material

We describe a patient who experienced dystonia coincident with the administration of an i.v. contrast agent. Dystonic reactions are not well known outside of the fields of Emergency Medicine, Neurology, and Psychiatry. They have not been previously reported as a reaction to i.v. contrast material. Prompt consideration and treatment of this condition may prevent unnecessary patient discomfort and interventions.