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Xie X, Guo J, Kong Y, Xie GX, Li L, Lv N, Xiao X, Tang J, Wang X, Liu P, Yang M, Xie Z, Wei W, Spencer DM, Xie X. Targeted expression of Escherichia coli purine nucleoside phosphorylase and Fludara® for prostate cancer therapy. J Gene Med 2013; 13:680-91. [PMID: 22009763 DOI: 10.1002/jgm.1620] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
Abstract
BACKGROUND Previous studies have shown that Herpes Simplex Virus thymidine kinase (HSV-tk)/ganciclovir (GCV) comprised the most commonly used suicide gene therapy for prostate cancer, with modest results being obtained. However, novel suicide genes, such as Escherichia coli purine nucleoside phosphorylase (PNP), have been utilized to demonstrate more potent tumor killing and an enhanced bystander effect on local, non-expressing cells compared to HSV-tk. METHODS PNP/fludarabine (Fludara®; fludarabine phosphate; Berlex Labs, Richmond, CA, USA) was deliveried by prostate-specific, rat probasin-based promoter, ARR2PB. After infection of various cell lines with ADV.ARR(2) PB-PNP and administration of androgen analog, R1881, expression of PNP mRNA was detected; in vivo, the antitumor effect of the ARR(2) PB-PNP/Fludara system was monitored and analyzed, as well as animal survival. RESULTS After in vitro infection with ADV.ARR(2) PB-PNP (multiplicity of infection = 10), LNCaP cells were more sensitive to a lower concentration Fludara (LD(50) , approximately 0.1 µg/ml) in the presence of R1881. Furthermore, robust bystander effects after R1881/Fludara treatment were observed in LNCaP cells after infection with bicistronic vector ADV.ARR2PB/PNP-IRES-EGFP in contrast to a much weaker effect in cells treated with ADV.CMV-HSV-tk/GCV. In vivo, tumor size in the ADV.ARR2PB-PNP/Fludara treatment group was dramatically smaller than in the control groups, and the mice treated with our system had a significantly prolonged survival, with three of eight mice surviving up to the 160-day termination point, as well as no systemic toxicity. CONCLUSIONS The ARR(2) PB-PNP/Fludara system induced massive tumor cell death and a prolonged life span without systemic cytotoxicity; therefore, it might be a more attractive strategy for suicide gene therapy of prostate cancer.
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Affiliation(s)
- Xinhua Xie
- State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China; Department of Breast Oncology, Sun Yat-Sen University Cancer Center, Guangzhou, China
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Jiang X, Ren Y, Williford JM, Li Z, Mao HQ. Liver-targeted gene delivery through retrograde intrabiliary infusion. Methods Mol Biol 2013; 948:275-284. [PMID: 23070777 DOI: 10.1007/978-1-62703-140-0_19] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/01/2023]
Abstract
Retrograde intrabiliary infusion (RII) has recently been characterized as a safe and effective administration route for liver-targeted gene delivery. Efficient transgene expression in the liver has been achieved by infusing a variety of gene vectors including adenovirus, retrovirus, lipoplexes, polyplexes, and naked DNA through the common bile duct. Here, we describe the RII technique and key infusion parameters for delivering plasmid DNA and DNA nanoparticles to the rat liver. After RII of plasmid DNA, the level of transgene expression in rat liver is comparable to that achieved by hydrodynamic injection of plasmid DNA, which is considered to be "gold standard" for liver-targeted gene delivery. RII has also been shown to significantly enhance the gene delivery efficiency by polymer/DNA nanoparticles in comparison with intravenous and intraportal infusions. This method induces minimal level of cytotoxicity and damage to the liver and bile duct. Due to these advantages, RII has the potential to be used for delivering various gene vectors in clinical setting through the endoscopic retrograde cholangiopancreatography procedure.
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Affiliation(s)
- Xuan Jiang
- Department of Materials Science and Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, USA
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Patil RR, Yu J, Banerjee SR, Ren Y, Leong D, Jiang X, Pomper M, Tsui B, Kraitchman DL, Mao HQ. Probing in vivo trafficking of polymer/DNA micellar nanoparticles using SPECT/CT imaging. Mol Ther 2011; 19:1626-35. [PMID: 21750533 DOI: 10.1038/mt.2011.128] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Successful translation of nonviral gene delivery to therapeutic applications requires detailed understanding of in vivo trafficking of the vehicles. This report compares the pharmacokinetic and biodistribution profiles of polyethylene glycol-b-polyphosphoramidate (PEG-b-PPA)/DNA micellar nanoparticles after administration through intravenous infusion, intrabiliary infusion, and hydrodynamic injection using single photon emission computed tomography/computed tomography (SPECT/CT) imaging. Nanoparticles were labeled with (111)In using an optimized protocol to retain their favorable physicochemical properties. Quantitative imaging analysis revealed different in vivo trafficking kinetics for PEG-b-PPA/DNA nanoparticles after different routes of administration. The intrabiliary infusion resulted in the highest liver uptake of micelles compared with the other two routes. Analysis of intrabiliary infusion by the two-compartment pharmacokinetic modeling revealed efficient retention of micelles in the liver and minimal micelle leakage from the liver to the blood stream. This study demonstrates the utility of SPECT/CT as an effective noninvasive imaging modality for the characterization of nanoparticle trafficking in vivo and confirms that intrabiliary infusion is an effective route for liver-targeted delivery of DNA-containing nanoparticles.
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Affiliation(s)
- Rajesh R Patil
- Department of Materials Science and Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA
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Lu ZZ, Zou XH, Dong LX, Qu JG, Song JD, Wang M, Guo L, Hung T. Novel recombinant adenovirus type 41 vector and its biological properties. J Gene Med 2009; 11:128-38. [PMID: 19097028 DOI: 10.1002/jgm.1284] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
BACKGROUND Human adenovirus serotype 41 (Ad41) is a natural pathogen of the digestive tract and can cause gastroenteritis. There has been interest in reconstructing Ad41 as a gene delivery vector targeting the gastrointestinal tract, which is hampered by its fastidiousness. METHODS An Ad41 E1B55K-transduced 293 cell line (293E12) was established as the packaging cell line. A backbone plasmid (pAdbone41) and a shuttle plasmid (pSh41-CMV) were constructed based on the Ad41 genome. Replication-defective adenovirus (Ad41-GFP) was rescued in 293E12 after being transfected with the linearized adenoviral plasmid, which was generated by homologous recombination of pAdbone41 and the shuttle plasmid carrying the GFP gene in Escherichia coli strain BJ5183. The packaging ability of 293E12, the stability of the Ad41-GFP genome and the acid-resistant property of Ad41-GFP were all investigated. RESULTS A 293E12 cell could produce approximately 9000 viral particles of Ad41-GFP, which is close to the amount in the control virus (Ad5-GFP) amplified in one 293 cell. Ad41-GFP contained a genetically stable genome after being passaged eight times in 293E12 cells. More significantly, Ad41-GFP was more resistant to acid exposure than Ad5-GFP. It retained almost complete viability when exposed to hydrochloric acid with a pH value of 2 for 30 min, whereas Ad5-GFP lost 99% of its viability under the same conditions. Ad41-GFP was also more tolerant to treatment with artificial digestive fluid. CONCLUSIONS An Ad41 vector system was successfully constructed, which consisted of the backbone plasmid, shuttle plasmid and packaging cell line 293E12. This system can be utilized to generate genetically stable and acid-resistant recombinant Ad41 carrying any gene of interest.
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Affiliation(s)
- Zhuo-Zhuang Lu
- National Institute for Viral Disease Control and Prevention, Chinese Centre for Disease Control and Prevention, Beijing, PR China
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Polyak S, Mah C, Porvasnik S, Herlihy JD, Campbell-Thompson M, Byrne BJ, Valentine JF. Gene delivery to intestinal epithelial cells in vitro and in vivo with recombinant adeno-associated virus types 1, 2 and 5. Dig Dis Sci 2008; 53:1261-70. [PMID: 17934813 PMCID: PMC3896329 DOI: 10.1007/s10620-007-9991-1] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/05/2007] [Accepted: 08/15/2007] [Indexed: 12/23/2022]
Abstract
Intestinal disorders such as inflammatory bowel disease (IBD) result in chronic illness requiring lifelong therapy. Our aim was to evaluate the efficacy of recombinant adeno-associated virus (AAV) vector-mediated gene delivery to intestinal epithelial cells in vitro and in vivo. Human colon epithelial cell lines and colon biopsies were transduced using AAV pseudotypes 2/1, 2/2, and 2/5 encoding green fluorescence protein (GFP). Mice were administered the same vectors through oral, enema, intraperitoneal (IP) injection and superior mesenteric artery (SMA) injection routes. Tropism and efficiency were determined by microscopy, flow cytometry, immunohistochemistry and PCR. Caco2 cells were more permissive to AAV transduction. Human colon epithelial cells in organ culture were more effectively transduced by AAV2/2. SMA injection provided the most effective means of vector gene transfer to small intestine and colonic epithelial cells in vivo. Transgene detection 80 days post AAV treatment suggests transduction of crypt progenitor cells. This study shows the feasibility of AAV-mediated intestinal gene delivery, applicable for the investigation of IBD pathogenesis and novel therapeutic options, but also revealed the need for further studies to identify more efficient pseudotypes.
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Affiliation(s)
- Steven Polyak
- Division of Gastroenterology, Department of Medicine, University of Florida, Gainesville, FL 32610, USA.
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Guan YS, La Z, Yang L, He Q, Li P. p53 gene in treatment of hepatic carcinoma: status quo. World J Gastroenterol 2007; 13:985-992. [PMID: 17373730 PMCID: PMC4146884 DOI: 10.3748/wjg.v13.i7.985] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/30/2006] [Revised: 12/12/2006] [Accepted: 01/16/2007] [Indexed: 02/06/2023] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the 10 most common cancers worldwide. There is no ideal treatment for HCC yet and many researchers are trying to improve the effects of treatment by changing therapeutic strategies. As the majority of human cancers seem to exhibit either abnormal p53 gene or disrupted p53 gene activation pathways, intervention to restore wild-type p53 (wt-p53) activities is an attractive anti-cancer therapy including HCC. Abnormalities of p53 are also considered a predisposition factor for hepatocarcinogenesis. p53 is frequently mutated in HCC. Most HCCs have defects in the p53-mediated apoptotic pathway although they carry wt-p53. High expression of p53 in vivo may exert therapeutic effects on HCC in two aspects: (1) High expression of exogenous p53 protein induces apoptosis of tumor cells by inhibiting proliferation of cells through several biologic pathways and (2) Exogenous p53 renders HCC more sensitive to some chemotherapeutic agents. Several approaches have been designed for the treatment of HCC via the p53 pathway by restoring the tumor suppression function from inactivation, rescuing the mutated p53 gene from instability, or delivering therapeutic exogenous p53. Products with p53 status as the target have been studied extensively in vitro and in vivo. This review elaborates some therapeutic mechanisms and advances in using recombinant human adenovirus p53 and oncolytic virus products for the treatment of HCC.
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Affiliation(s)
- Yong-Song Guan
- Department of Radiology and Oncology, West China Hospital of Sichuan University, Chengdu 610041, Sichuan Province, China.
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Jiang X, Dai H, Leong KW, Goh SH, Mao HQ, Yang YY. Chitosan-g-PEG/DNA complexes deliver gene to the rat liver via intrabiliary and intraportal infusions. J Gene Med 2006; 8:477-87. [PMID: 16389625 DOI: 10.1002/jgm.868] [Citation(s) in RCA: 93] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
BACKGROUND Chitosan has been shown to be a non-toxic and efficient vector for in vitro gene transfection and in vivo gene delivery through pulmonary and oral administrations. Recently, we have shown that chitosan/DNA nanoparticles could mediate high levels of gene expression following intrabiliary infusion 1. In this study, we have examined the possibility of using polyethylene glycol (PEG)-grafted chitosan/DNA complexes to deliver genes to the liver through bile duct and portal vein infusions. METHODS PEG (Mw: 5 kDa) was grafted onto chitosan (Mw: 47 kDa, deacetylation degree: 94%) with grafting degrees of 3.6% and 9.6% (molar percentage of chitosan monosaccharide units grafted with PEG). The stability of chitosan-g-PEG/DNA complexes was studied by measuring the change in particle size and by agarose gel electrophoresis against bile or serum challenge. The influence of PEG grafting on gene transfection efficiency was evaluated in HepG2 cells using luciferase reporter gene. Chitosan and chitosan-g-PEG/DNA complexes were delivered to the liver through bile duct and portal vein infusions with a syringe pump. Gene expression in the liver and the distribution of gene expression in other organs were evaluated. The acute liver toxicity of chitosan and chitosan-g-PEG/DNA complexes was examined by measuring serum alanine aminotranferase (ALT) and aspartate aminotransferase (AST) activities as a function of time. RESULTS Both chitosan and chitosan-g-PEG displayed comparable gene transfection efficiency in HepG2 cells. After challenge with serum and bile, chitosan-g-PEG/DNA complexes, especially those prepared with chitosan-g-PEG (GD = 9.6%), did not form large aggregates like chitosan/DNA complexes but remained stable for up to 30 min. In addition, chitosan-g-PEG prevented the degradation of DNA in the presence of serum and bile. On day 3 after bile duct infusion, chitosan-g-PEG (GD = 9.6%)/DNA complexes mediated three times higher gene expression in the liver than chitosan/DNA complexes and yielded background levels of gene expression in other organs. On day 1 following portal vein infusion, gene expression level induced by chitosan/DNA complexes was hardly detectable but chitosan-g-PEG (GD = 9.6%) mediated significant transgene expression. Interestingly, transgene expression by chitosan-g-PEG/DNA complexes in other organs after portal vein infusion increased with increasing grafting degree of PEG. The ALT and AST assays indicated that grafting of PEG to chitosan reduced the acute liver toxicity towards the complexes. CONCLUSION This study demonstrated the potential of chitosan-g-PEG as a safe and more stable gene carrier to the liver.
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Affiliation(s)
- Xuan Jiang
- Institute of Bioengineering and Nanotechnology, Singapore 138669, Singapore
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Cheng X, Ming X, Croyle MA. PEGylated adenoviruses for gene delivery to the intestinal epithelium by the oral route. Pharm Res 2004; 20:1444-51. [PMID: 14567640 DOI: 10.1023/a:1025714412337] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
PURPOSE Adenoviruses are being developed for diseases of the gastrointestinal tract. Several in vitro assays were used to predict stability of PEGylated adenovirus along the GI tract and determine in vivo gene transfer after oral administration. METHODS Recombinant adenovirus was modified with monomethoxypoly(ethylene) glycols activated by cyanuric chloride, succinimidyl succinate, and tresyl chloride. Transduction efficiency was assessed on Caco-2 cells. In vitro stability of viruses in simulated gastric fluid, pancreatic fluid, and bile was assessed by serial dilution on 293 cells. Transduction efficiency in vivo was determined by oral administration of 1 x 10(12) particles of unmodified or PEGylated virus to fasted Sprague-Dawley rats. RESULTS Titers of unmodified virus declined to undetectable levels after 40 min in simulated gastric fluid while the infectious titer of the modified vectors did not change for 3 h. Similar results were seen with simulated pancreatic fluid. PEGylation also enhanced adenoviral transduction efficiency in Caco-2 cells by a factor of 20. PEGylation enhanced adenovirus transduction efficiency 10- to 40-fold in vivo in intestinal segments that do not express significant amounts of adenovirus receptors (jejunum, colon) with transgene expression located in the crypt regions. CONCLUSIONS PEGylated adenoviruses are suitable gene delivery vehicles for oral administration.
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Affiliation(s)
- Xuan Cheng
- College of Pharmacy, Division of Pharmaceutics, The University of Texas at Austin, Austin, Texas 78712, USA
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Zhang X, Collins L, Sawyer GJ, Dong X, Qiu Y, Fabre JW. In vivo gene delivery via portal vein and bile duct to individual lobes of the rat liver using a polylysine-based nonviral DNA vector in combination with chloroquine. Hum Gene Ther 2001; 12:2179-90. [PMID: 11779402 DOI: 10.1089/10430340152710522] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
The objective of this study was to evaluate a bifunctional synthetic peptide as a DNA vector for regional gene delivery to the rat liver by the portal vein and bile duct routes. The 31-amino-acid peptide (polylysine-molossin) comprises an amino-terminal chain of 16 lysines for electrostatic binding of DNA, and the 15 amino acid integrin-binding domain of the venom of the American pit viper, Crotalus molossus molossus. Initial in vitro evaluation demonstrated that polylysine-molossin/DNA complexes were much smaller (approximately 50-100nm versus 500-1300nm), more positively charged, and more stable in isotonic dextrose in comparisons with salt-containing solutions. However, polylysine-molossin/DNA complexes in any solution other than complete culture medium were ineffective for gene delivery in vitro. Vector localization studies demonstrated that both the portal vein and bile duct routes provided excellent access of polylysine-molossin/DNA complexes to the liver. However, complexes delivered by the portal vein were rapidly lost (<15 min) following re-establishment of the portal circulation, whereas complexes delivered by the bile duct persisted much longer. Polylysine-molossin/DNA complexes in various isotonic solutions were delivered to the right lateral lobes either by perfusion through a branch of the portal vein or by infusion into appropriate branches of the bile duct. Two or three hours before gene delivery, rats were given a single injection of chloroquine. We report that the polylysine-molossin vector is much more effective (>10-fold) when delivered by the bile duct route with all isotonic solutions evaluated, and that polylysine-molossin/DNA complexes in isotonic dextrose are much more effective (>10-fold) than complexes in salt-containing solutions.
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Affiliation(s)
- X Zhang
- Department of Clinical Sciences, Institute of Liver Studies, Guy's, King's and St. Thomas' School of Medicine, King's College Hospital, Bessemer Road, London SE5 9PJ, UK
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Xie X, Zhao X, Liu Y, Young CY, Tindall DJ, Slawin KM, Spencer DM. Robust prostate-specific expression for targeted gene therapy based on the human kallikrein 2 promoter. Hum Gene Ther 2001; 12:549-61. [PMID: 11268287 DOI: 10.1089/104303401300042483] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Tissue-specific transcriptional regulatory elements can increase the safety of gene therapy vectors. Unlike prostate-specific antigen (PSA/hK3), whose expression displays an inverse correlation with prostate cancer grade and stage, human glandular kallikrein 2 (hK2) is upregulated in higher grade and stage disease. Therefore, our goal was to develop a strong and prostate-specific hK2-based promoter for targeted gene therapy. We identified the minimum "full-strength" hK2 enhancer and built transcriptional regulatory elements composed of multiple tandem copies of this 1.2-kb enhancer, fused to the hK2 minimal promoter. Relative to the weak induction of the minimal hK2 promoter by androgen analog (R1881) in androgen receptor (AR)-positive LNCaP cells, transcriptional activity was increased by 25-, 44-, 81-, and 114-fold when one to four enhancers were spliced to the hK2 promoter, respectively. In contrast, the enhancer/promoter elements were inactive in the AR(-) prostate cancer line PC-3 and in a panel of nonprostate lines, including 293, U87, MCF-7, HuH-7, and HeLa cells. Furthermore, we generated a recombinant adenovirus, ADV.hK2-E3/P-EGFP, expressing enhanced green fluorescent protein (EGFP) under the control of the hK2 triplicate enhancer/promoter, and compared its properties with ADV.CMV-EGFP expressing EGFP under the control of the cytomegalovirus (CMV) enhancer/promoter. Unlike the CMV promoter, the hK2-E3/P promoter was at least 100-fold inducible by R1881 in the adenoviral backbone. Compared with in situ injection of subcutaneous LNCaP tumors with ADV.CMV-EGFP, which led to detectable EGFP expression in tumor, liver, and brain tissue, ADV.hK2-E3/P-EGFP injection led to robust but tumor-restricted EGFP expression. These results suggest that the hk2 multienhancer/promoter should be a powerful novel reagent for safer targeted gene therapy of prostate cancer.
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MESH Headings
- Adenoviridae/genetics
- Animals
- Binding Sites
- Brain/metabolism
- Cytomegalovirus/genetics
- Dose-Response Relationship, Drug
- Enhancer Elements, Genetic
- Flow Cytometry
- Genetic Therapy/methods
- Genetic Vectors/metabolism
- Green Fluorescent Proteins
- HeLa Cells
- Humans
- Liver/metabolism
- Luminescent Proteins/metabolism
- Male
- Mice
- Mice, Inbred BALB C
- Mice, Nude
- Microscopy, Fluorescence
- Models, Genetic
- Neoplasm Transplantation
- Plasmids/metabolism
- Promoter Regions, Genetic
- Prostate/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Spectrometry, Fluorescence
- Tissue Kallikreins/biosynthesis
- Tissue Kallikreins/genetics
- Transcription, Genetic
- Transduction, Genetic
- Transfection
- Tumor Cells, Cultured
- Up-Regulation
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Affiliation(s)
- X Xie
- Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA
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