bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intramuscular SP1017‐formulated DNA electrotransfer enhances transgene expression and distributes hHGF to different rat tissues
Authors: Riera Marta , Chillon Miguel , Aran Josep M. , Cruzado Josep M. , Torras Joan , Grinyó Josep M. , Fillat Cristina 
Published: January 2004

AbstractBackgroundThe systemic administration of a therapeutic protein is a common approach for the treatment of multiple disorders. Intramuscular (i.m.) injection of plasmids, followed by electroporation, has been shown to facilitate naked DNA gene transfer in skeletal muscle allowing proteins to be produced and secreted at therapeutically relevant levels.MethodsPlasmid DNA, unformulated or formulated with the non‐ionic carrier SP1017, was injected at the rat tibialis anterior muscle followed by the application of electric pulses. Follow‐up of protein expression was measured.ResultsIn our study we report that the non‐ionic carrier SP1017 significantly increases transgene expression in rat muscle after the i.m. injection of a formulated‐pCMVβ plasmid followed by electroporation. Such increased expression was observed after delivering square‐wave unipolar electric pulses at two different field strengths: low (110 V/cm) and high (175 V/cm). Moreover, elevated secreted placental alkaline phosphatase (SEAP) plasma levels were achieved with low‐voltage (110 V/cm) electroporation. Our results also show that human hepatocyte growth factor (hHGF) can be produced from rat muscle and delivered to blood circulation at a biologically active level after a single i.m. injection of an SP1017‐formulated plasmid (pCMV/hHGF) followed by electroporation. Tissue distribution studies mostly identified hHGF in the liver, but it was also found in the kidneys and lungs suggesting that here too the HGF could be of therapeutic benefit.ConclusionsOur results indicate that SP1017 enhances the expression of electrotransferred genes. Such a delivery approach could prove an efficient method for the systemic production of therapeutic proteins. Copyright © 2003 John Wiley & Sons, Ltd.
Local RAD50 gene delivery induces regression of preformed porcine coronary in‐stent neointimal hyperplasia
Authors: Ahn Young Keun , Kook Hyun , Jeong Myung Ho , Ahn Kyu Youn , Cho Jeong Gwan , Park Jong Chun , Kang Jung Chaee , Kim Kyung Keun 
Published: January 2004

AbstractBackgroundRecently, we observed that overexpression of human RAD50 (hRAD50) induced p21‐dependent cytotoxicity in various cultured cells, and rat and mouse tumor models. This study investigated the characteristics of endothelial cell (EC) death by hRAD50 and the potential utility of hRAD50 in the development of gene therapies for vascular restenosis.MethodsWe studied the effects of transient hRAD50 gene transfer using nonliposomal lipid on the survival of primary cultured human coronary arterial EC and smooth muscle cells (SMC). Palmaz‐Schatz stents were deployed in two epicardial coronary arteries in each pig (n = 10). Two weeks later, the patency of the stented arteries was documented by coronary angiography, and the hRAD50 construct or empty vector mixed with lipid was delivered to one of the stented arteries in each pig using a Dispatch catheter. Coronary angiography was repeated 2 weeks after gene delivery and histological examination was performed.ResultsLipid‐mediated hRAD50 gene transfer resulted in the death of EC and SMC. It also increased endothelial nitric oxide synthase (eNOS) expression and nitrite production as well as p21 expression. Pretreatment with NOS and pan‐caspase inhibitors completely prevented EC death by hRAD50. In the hRAD50‐delivered arteries, the percentage of diameter stenosis, neointimal area, and pathologic area of stenosis were significantly smaller than in the control arteries. eNOS expression increased in the hRAD50‐delivered arteries. Systemic hematologic and chemical values were not affected by gene delivery.ConclusionsSignificant regression of preformed in‐stent neointimal hyperplasia was induced by local hRAD50 gene delivery to stented porcine coronary arteries without apparent systemic toxicity. Copyright © 2003 John Wiley & Sons, Ltd.
In vivo expansion of gene‐modified hematopoietic cells by a novel selective amplifier gene utilizing the erythropoietin receptor as a molecular switch
Authors: Nagashima Takeyuki , Ueda Yasuji , Hanazono Yutaka , Kume Akihiro , Shibata Hiroaki , Ageyama Naohide , Terao Keiji , Ozawa Keiya , Hasegawa Mamoru 
Published: January 2004

AbstractBackgroundIn vivo expansion of gene‐modified cells would be a promising approach in the field of hematopoietic stem cell gene therapy. To this end, we previously developed a selective amplifier gene (SAG), a chimeric gene encoding the granulocyte colony‐stimulating factor (G‐CSF) receptor (GCR), as a growth‐signal generator and the hormone‐binding domain of the steroid receptor as a molecular switch. We have already reported that hematopoietic cells retrovirally transduced with the SAG can be expanded in a steroid‐dependent manner in vitro and in vivo in mice and nonhuman primates. In this study, we have developed a new‐generation SAG, in which the erythropoietin (EPO) receptor (EPOR) is utilized instead of the steroid receptor as a molecular switch.MethodsTwo EPO‐driven SAGs were constructed, EPORGCR and EPORMpl, containing the GCR and c‐Mpl as a signal generator, respectively. First, to compare the steroid‐driven and EPO‐driven SAGs, Ba/F3 cells were transduced with these SAGs. Next, to examine whether GCR or c‐Mpl is the more suitable signal generator of the EPO‐driven SAG, human cord blood CD34+ cells were transduced with the two EPO‐driven SAGs (EPORMpl and EPORGCR). Finally, we examined the in vivo efficacy of EPORMpl in mice. Irradiated mice were transplanted with EPORMpl‐transduced bone marrow cells followed by administration of EPO.ResultsThe EPO‐driven SAGs were shown to induce more rapid and potent proliferation of Ba/F3 cells than the steroid‐driven SAGs. The EPORMpl induced more efficient EPO‐dependent proliferation of the human cord blood CD34+ cells than the EPORGCR in terms of total CD34+ cell, c‐Kit+ cell, and clonogenic progenitor cell (CFU‐C) numbers. In the transplanted mice the transduced peripheral blood cells significantly increased in response to EPO.ConclusionsThe new‐generation SAGs, especially EPORMpl, are able to efficiently confer an EPO‐dependent growth advantage on transduced hematopoietic cells in vitro and in vivo in mice. Copyright © 2003 John Wiley & Sons, Ltd.
Partial correction of the α‐galactosidase A deficiency and reduction of glycolipid storage in Fabry mice using synthetic vectors
Authors: Przybylska Malgorzata , Wu I‐Huan , Zhao Hongmei , Ziegler Robin J. , Tousignant Jennifer D. , Desnick Robert J. , Scheule Ronald K. , Cheng Seng H. , Yew Nelson S. 
Published: January 2004

AbstractBackgroundFabry disease is a recessive, X‐linked disorder caused by a deficiency of the lysosomal enzyme α‐galactosidase A, leading to an accumulation of the glycosphingolipid globotriaosylceramide (GL‐3) in most tissues of the body. The goal of this study was to determine if systemic delivery of a nonviral vector could correct the enzyme deficiency and reduce the levels of GL‐3 in different tissues of a transgenic knockout mouse model of the disease.MethodsCationic lipid was complexed with a CpG‐depleted plasmid DNA vector and then injected intravenously into Fabry mice. The levels of α‐galactosidase A and GL‐3 in different tissues were assayed at various time points after injection.ResultsExpression of α‐galactosidase A was detected in the different tissues of Fabry mice for up to 3 months after complex administration, but resulted in minimal reductions in GL‐3 levels. However, the use of the anti‐inflammatory drug dexamethasone and multiple dosing increased α‐galactosidase A expression and resulted in significant reductions of GL‐3 in all the organs with the exception of the kidney. In addition, injecting complex into young Fabry mice partially prevented the normal accumulation of GL‐3 in the heart, lung, and liver.ConclusionsSystemic delivery of a cationic lipid–pDNA complex partially corrected the enzyme deficiency and reduced glycolipid storage in a mouse model of Fabry disease. The results are one of the few demonstrations of long‐term efficacy in a genetic disease model using nonviral vectors. However, substantial improvements in expression, especially in critical organs such as the kidney, are required before these vectors can become a viable approach to treat Fabry disease and other lysosomal storage disorders. Copyright © 2003 John Wiley & Sons, Ltd.
Factors influencing immune response after in vivo retrovirus‐mediated gene transfer to the liver
Authors: Podevin Guillaume , Otta Edson , Nguyen Jean Michel , Pichard Virginie , Aubert Dominique , Moullier Philippe , Ferry Nicolas 
Published: January 2004

AbstractBackgroundHighly efficient retrovirus‐mediated gene transfer into hepatocytes in vivo triggers an immune response directed against transduced hepatocytes. This effect may be due either to spreading of retroviral vectors in the blood stream with subsequent infection of antigen presenting cells (APCs) or to cross‐presentation of the transgene product present as a contaminant in the viral stock. In order to decrease immune response, we evaluated the effect of asanguineous perfusion of the liver as well as purification of the viral stock on long‐term transduction of hepatocytes using the nls‐lacZ marker gene.MethodsAnimals were divided in four groups. In group 1, the viral supernatant was perfused in the regenerating liver after complete vascular exclusion of the organ. In group 2, using the same strategy, animals received retroviral supernatant that was passed through a β‐galactosidase affinity column to reduce β‐galactosidase contamination. In two control groups (respectively groups 3 and 4) the corresponding viral supernatants were delivered via peripheral injection.ResultsIn group 1, 23.1% of animals had no immune response 2 months after gene delivery vs. 33.4% in group 2, 4.3% in control group 3, and 0% in control group 4. Statistical analysis of the results demonstrated that only the difference between groups 2 and 3 was statistically significant. This indicated that both asanguineous perfusion together with passage through an affinity column were required to decrease significantly immune response.ConclusionsOur present results suggest that both supernatant contamination and viral spreading contribute to immune response after retrovirus‐mediated gene delivery to the liver. Copyright © 2003 John Wiley & Sons, Ltd.
In situ single cell observation by fluorescence resonance energy transfer reveals fast intra‐cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine
Authors: Itaka Keiji , Harada Atsushi , Yamasaki Yuichi , Nakamura Kozo , Kawaguchi Hiroshi , Kataoka Kazunori 
Published: January 2004

AbstractBackgroundThe investigation into the intracellular mechanisms for gene expression has acquired great impetus for the improvement of the transfection efficiency by a non‐viral gene delivery system.MethodsIntracellular trafficking as well as release of plasmid DNA (pDNA) complexed with polycations, including linear and branched polyethylenimine (LPEI, BPEI) and poly(L‐lysine) (PLL), were explored under confocal microscopy using fluorescence resonance energy transfer (FRET) between a pair of donor–acceptor fluorescent dyes (fluorescein and Cy3) tagged on a single pDNA molecule.ResultspDNA complexed with LPEI underwent a rapid escape from the endosomes, spreading uniformly into the cytoplasm with a substantial decrease in FRET efficiency due to the disintegration of LPEI/pDNA polyplex structure. pDNA complexed with BPEI also achieved a rapid escape from the endosomes. Nevertheless, the pDNA retained high FRET efficiency even after 24 h, indicating an appreciable stability of the BPEI/pDNA polyplex to keep pDNA in a condensed state. In the PLL/pDNA polyplexes, neither endosome escape nor pDNA decondensation was observed. These intracellular characteristics of polyplexes showed a clear correlation to their gene transfection efficiency: The LPEI/pDNA revealed a considerably higher and faster gene expression compared with BPEI/pDNA. Atomic force microscopy revealed that BPEI induced more effective condensation of pDNA than LPEI, being consistent with restricted cytoplasmic release of complexed pDNA.ConclusionFast endosomal escape and subsequent smooth disintegration of LPEI/pDNA in the cytoplasm are likely to be determining factors for the excellent transfection efficiency of this polyplex system. These properties may be particularly beneficial to achieve appreciably high gene expression in a prompt manner. Copyright © 2003 John Wiley & Sons, Ltd.
Use of the norepinephrine transporter as a reporter gene for non‐invasive imaging of genetically modified cells
Authors: Anton Martina , Wagner Bettina , Haubner Roland , Bodenstein Claudia , Essien Bryan E. , Bönisch Heinz , Schwaiger Markus , Gansbacher Bernd , Weber Wolfgang A. 
Published: January 2004

AbstractBackgroundThe norepinephrine transporter (NET) is a high‐affinity transporter for catecholamines. Its expression is almost exclusively restricted to the sympathetic nervous system. In this study we evaluated whether the NET can be used as a reporter gene for non‐invasive imaging of genetically modified cells with radiolabeled probes.MethodsHuman A431, HT1080 and murine CMS‐5 cells were retrovirally transduced with bovine NET cDNA. Transduced and parental cells were incubated in vitro with [131I]meta‐iodobenzylguanidine ([131I]MIBG). The specificity of tracer uptake was determined by adding the NET inhibitor imipramine. Rat PC12 cells served as positive controls. Parental and A431NET cells were xenotransplanted into nude mice and tumor uptake of [123I]MIBG in vivo was determined after tracer administration.ResultsIn vitro stably transduced cells showed a 66‐ to 120‐fold higher [131I]MIBG uptake than parental cells. Incubation with imipramine reduced [131I]MIBG uptake of transduced cells to the level found in parental cells. More than 70% of the initial radioactivity was retained in all transduced cell lines after 2 h incubation with tracer‐free medium. [131I]MIBG uptake in PC12 cells, which express the NET endogenously, was 20‐ to 28‐fold lower than in transduced cells. In vivo, A431NET tumors demonstrated a 33‐fold higher [123I]MIBG uptake than parental tumors. Gamma camera images 24 h after tracer injection showed no tracer uptake in parental A431 tumors, but clear images of A431NET tumors.ConclusionsTransduction of tumor cells with NET cDNA causes highly specific uptake and significant retention of catecholamine analogs in vitro and in vivo. These characteristics make the NET suitable as a reporter gene for non‐invasive monitoring of gene transfer. Copyright © 2003 John Wiley & Sons, Ltd.
Gene‐directed enzyme prodrug therapy for prostate cancer in a mouse model that imitates the development of human disease
Authors: Martiniello‐Wilks Rosetta , Dane Allison , Voeks Dale J. , Jeyakumar Geetha , Mortensen Elin , Shaw Jan M. , Wang Xiao Yang , Both Gerald W. , Russell Pamela J. 
Published: January 2004

AbstractBackgroundGene‐directed enzyme prodrug therapy (GDEPT) based on the E. coli enzyme purine nucleoside phosphorylase (PNP) represents a new approach for treating slow growing tumours like prostate cancer (PCa). Expressed enzyme converts a systemically administered prodrug, fludarabine phosphate, to a toxic metabolite, 2‐fluoroadenine. Infected and neighbouring cells are killed by a bystander effect that results from the inhibition of DNA and RNA synthesis.MethodsThese studies were carried out using the transgenic adenocarcinoma of the prostate (TRAMP) model that mimics human PCa development and progression. Control TRAMP mice were injected intraprostatically with vector vehicle and thereafter intraperitoneally with saline or fludarabine phosphate (∼600 mg/m2/day) once daily for 5 consecutive days. Treated mice received a single intraprostatic injection containing 1010 particles of OAdV220, an ovine atadenovirus which expresses the E. coli PNP gene under the control of the Rous sarcoma virus promoter, followed by systemic fludarabine treatment. The weight of the genitourinary tract, seminal vesicles and the prostate as well as animal survival were monitored. Tumours were also analysed histologically.ResultsPreliminary studies showed that fludarabine alone caused no significant change in genitourinary (GU) tract weight in TRAMP mice. Animals injected with vector and prodrug showed a significant reduction (36–47%) in GU tract weight (ANOVA p = 0.0002) and a 35–50% reduction in seminal vesicle weight (ANOVA p = 0.0007). In particular, the target organ showed a significant 57% reduction in prostate weight (ANOVA p = 0.0007). PNP‐GDEPT mice also showed a survival advantage over control mice. Histological analysis suggested that the cancer progression was slowed in GDEPT‐treated animals.ConclusionA single course of GDEPT based on OAdV‐delivered PNP and fludarabine produced highly significant suppression of PCa progression in immune‐competent TRAMP mice. Copyright © 2004 John Wiley & Sons, Ltd.
Steric stabilization of poly(2‐(dimethylamino)ethyl methacrylate)‐based polyplexes mediates prolonged circulation and tumor targeting in mice
Authors: Verbaan F. J. , Oussoren C. , Snel C. J. , Crommelin D. J. A. , Hennink W. E. , Storm G. 
Published: January 2004

AbstractBackgroundEfficient tumor targeting of polymeric gene transfer systems (polyplexes) represents a major challenge. To establish tumor targeting after intravenous (IV) administration, the circulation lifetime of these systems should be sufficiently long. Since naked polyplexes are rapidly eliminated from the circulation after IV adminstration, strategies have to be developed to improve their pharmacokinetics.MethodsComplexes of plasmid DNA and poly(2‐(dimethylamino)ethyl methacrylate) (pDMAEMA)‐graft‐PEG or AB di‐block copolymers of pDMAEMA and PEG, as well as PEGylated complexes prepared via PEGylation of preformed complexes (postPEGylation), were evaluated for their physicochemical properties (size and charge) their interactions with blood constituents and transfection activity in vitro. The pharmacokinetics and biodistribution of PEG‐polyplexes were studied in mice after IV administration. The degree of accumulation in two subcutaneous (SC) mouse tumors after IV administration was evaluated for the system with the longest circulation time.ResultsIt is shown that the surface charge of the pDMAEMA‐polyplexes was effectively shielded by two PEGylation methods (i.e. the use of pDMAEMA‐graft‐PEG polymers and postPEGylation). The shielding effect was the highest for the postPEGylation method with PEG20000, yielding polyplexes that hardly show interactions with blood components (i.e. albumin and erythrocytes) and show substantially prolonged circulation time in mice after IV administration. The superior colloidal stability and circulation kinetics of the postPEGylated polyplexes translated into tumor accumulation which amounted to about 3.5% of the injected dose per gram tumor tissue in a SC Neuro2A tumor model and to about 4.2% of the injected dose per gram tumor tissue in a SC C26 tumor model.ConclusionsThis study shows that postPEGylation of pDMAEMA‐based polyplexes is the most attractive method to prepare polyplexes with long circulating properties. Tumor targeting capacity after intravenous administration was demonstrated in two subcutaneous tumor models. Copyright © 2004 John Wiley & Sons, Ltd.
Developing a convenient large animal model for gene transfer to salivary glands in vivo
Authors: Li Jun , Zheng Chanyu , Zhang Xin , Liu Xiaoyong , Zhang Chengmei , Goldsmith Corinne M. , Baum Bruce J. , Wang Songlin 
Published: January 2004

AbstractBackgroundLocalized gene transfer to salivary glands has great potential for the treatment of salivary gland, systemic, and oral diseases. The minipig parotid gland, given its volume and morphological similarities to the human parotid gland, may be useful as a large animal model for pre‐clinical gene transfer experiments. The purpose of this study was to perform an initial assessment of the efficacy and safety of adenoviral‐vector‐mediated gene transfer to parotid glands of miniature pigs.MethodsAdCMVluc, a recombinant type 5 adenoviral (rAd5) vector containing a luciferase reporter gene, was administered to miniature pig parotid glands by intraductal cannulation. Five regions of gland tissue were obtained to measure the distribution of luciferase activity. The effects of time, viral dose, infusate buffer volume, and gland anatomical region on transgene expression were determined. Detailed serum chemistry and hematological analyses were performed. In addition, AdCMVlacZ, a similar rAd5 vector encoding β‐galactosidase, was also delivered to determine the parotid gland cell types transduced.ResultsLuciferase assays indicated that gene transfer to miniature pig salivary glands could be readily accomplished using rAd5 vectors. Highest transgene expression was found in the center of glands, which was > posterior > inferior > anterior > superior tissue regions. Expression was maximal on day 2 and declined to background by day 14, and observed in both acinar and ductal cells. Several serum chemistry and hematology parameters were transiently changed following rAd5 administration.ConclusionsTransgene expression by, and inflammatory response to, rAd5 vectors in minipig parotid glands are similar to results seen earlier in rodent studies. This suggests that results of salivary gland gene transfer from rodent studies can be extended to a larger animal model, and supports the value of using minipigs for pre‐clinical applications of gene transfer to these tissues. Published in 2004 by John Wiley & Sons, Ltd.
Local ex vivo gene therapy with bone marrow stromal cells expressing human BMP4 promotes endosteal bone formation in mice
Authors: Zhang Xiao S. , Linkhart Thomas A. , Chen Shin‐Tai , Peng Hairong , Wergedal Jon E. , Guttierez Genevieve G. , Sheng Matilda H.‐C. , William Lau K.‐H. , Baylink David J. 
Published: January 2004

AbstractBackgroundBone loss in osteoporosis is caused by an imbalance between resorption and formation on endosteal surfaces of trabecular and cortical bone. We investigated the feasibility of increasing endosteal bone formation in mice by ex vivo gene therapy with bone marrow stromal cells (MSCs) transduced with a MLV‐based retroviral vector to express human bone morphogenetic protein 4 (BMP4).MethodsWe assessed two approaches for administering transduced MSCs. β‐Galactosidase (β‐Gal) transduced C57BL/6J mouse MSCs were injected intravenously via tail vein or directly injected into the femoral bone marrow cavity of non‐marrow‐ablated syngenic recipient mice and bone marrow cavity engraftment was assessed. BMP4‐ or β‐Gal‐transduced cells were injected into the femoral bone marrow cavity and effects on bone were evaluated by X‐ray, peripheral quantitative computed tomography (pQCT), and histology.ResultsAfter tail‐vein injection less than 20% of recipient mice contained β‐Gal‐positive donor cells in femur, humerus or vertebra marrow cavities combined, and in these mice only 0.02–0.29% of injected cells were present in the bone marrow. In contrast, direct intramedullary injection was always successful and an average of 2% of injected cells were present in the injected femur marrow cavity 24 hours after injection. Numbers of donor cells decreased over the next 14 days. Intramedullary injection of BMP4‐transduced MSCs induced bone formation. Trabecular bone mineral density (BMD) determined by pQCT increased 20.5% at 14 days and total BMD increased 6.5% at 14 days and 10.4% at 56 days.ConclusionsThe present findings support the feasibility of using ex vivo MSC‐based retroviral gene therapy to induce relatively sustained new bone formation, with normal histological appearance, at endosteal bone sites. Copyright © 2004 John Wiley & Sons, Ltd.