Lentiviruses can infect mitotic and non-dividing cells owing to the karyophilic properties of their pre-integrating complex, which allow its active import through the nucleopore. Thus lentiviral vectors derived from human immunodeficiency virus type 1 can mediate an efficient transfer integration, and stable expression of transgenes into proliferating and stationary cells both in vivo and in vitro. By adopting the internal ribosome entry site of encephalomyocarditis virus for bicistronic expression or two promoters of EF-1alpha and SV40 for separate expression of two genes of interest, we developed two lentiviral vectors that express two genes. On FACS analysis, RT-PCR, and immunofluorescence assay, it was shown that the target cells expressed two genes of interest at different levels as the transducing vectors designed for. This vector system is useful especially for a stable, dual-gene expression and two transgene deliveries to non-dividing cells.
Encephalomyocarditis virus ; Fluorescent Antibody Technique ; HIV-1 ; Lentivirus ; Peptide Elongation Factor 1 ; Ribosomes ; Transgenes*

OBJECTIVETo investigate the genetic stability during the serial passages of non-replicating recombinant adenoviruses based on the novel AdEasy system.

METHODSFour non-replicating adenoviruses expressing rotavirus antigens, which were generated by the AdEasy system, were used as models and continuously propagated on 293 cells for 20 passages. Samples of the infected cells were collected at every 5 passages for the PCR analysis of the inserted rotavirus genes, replication-competent adenoviruses (RCAs) as well as the Western blot examination of the expression of the certain rotavirus genes.

RESULTSThe adenoviruses can be stably propagated on 293 cells. The existence and the stable expression of inserted rotavirus genes were able to be detected during the serial passage. The RCAs were not found within the first 10 passages, but the rvAdG2VP7(o) at passage 15 and the rvAdG2VP7(o) and rvAdG1VP7(o) at passage 20.

CONCLUSIONThe non-replicating adenoviruses showed promising genetic stability during the continuous passage on 293 cells. The RCAs normally will not be detectable within 10 continuous passages. The results indicated the potential of such recombinant adenoviruses in the research and development of the gene therapies and adenoviral-vectored vaccines.

Adenoviridae ; genetics ; isolation & purification ; physiology ; Cell Line ; DNA, Recombinant ; genetics ; Humans ; Serial Passage ; Transgenes ; Virus Replication

Animal models of Alzheimer disease (AD) are used to study the mechanisms underlying AD pathogenesis, genetic interactions with genes of interest, and environmental risk factors that cause sporadic AD as well as to test the therapeutic effects of AD drug-candidates on neuropathology and cognitive function. To attain a comparative view on the AD models developed, representative AD lines were selected and summarized with respect to transgenic constructs and AD-related pathology. In addition, age-dependent plaque deposition data available in the literature for six representative AD models such as Tg2576, PDAPP, TgAPP23, Tg-APPswe/PS1dE9, 3xTg-AD, and 5XFAD mice were reevaluated using a photographic plaque reference scale method that was introduced recently. Tg2576, PDAPP, and TgAPP23 mice, which carry the amyloid precursor protein (APP) transgene, produced initially slow, but progressively accelerated plaque deposition as they aged, resulting in logistic plaque deposition. In contrast, Tg-APPswe/PS1dE9 and 3xTg-AD mice, which carry both APP and PS1 transgenes, developed abruptly accelerated plaque formation from the beginning, resulting in logarithmic plaque deposition. 5XFAD mice, which also carry both the APP and PS1 transgenes, developed a logarithmic deposition beginning at 2 months. This comparative analysis suggests that AD models may be classified into two distinct plaque deposition groups, and that early plaque models such as APPswe/PS1dE9, 3xTg-AD and 5XFAD might be useful to study the biochemical aspects of APP metabolism, whereas late plaque models such as Tg2576, PDAPP, and TgAPP23 might be useful to study more physiological and environmental aspects of AD pathogenesis, which occur on a longer time scale.
Aged ; Alzheimer Disease ; Amyloid ; Animals ; Humans ; Mice ; Models, Animal ; Risk Factors ; Transgenes

BACKGROUND AND OBJECTIVES: Compared to other target cells examined for gene therapy, vascular smooth muscle cells (VSMCs) have the unique advantages including proximity to blood stream and relative abundance in vasculature. With an ultimate goal of developing VSMC-based therapies for cardiovascular disorders, we explored the utility of VSMC as a target cell for ex vivo gene therapy using a set of retroviral vectors. MATERIALS AND METHODS: Cultured VSMCs were transduced with replication-defective recombinant retroviruses harboring LacZ, nlsLacZ, mVEGF, mGM-CSF or bacterial CAT reporter. The VSMCs were examined for G418-selection, transduction efficiency, the level of transgene expression, and longevity of gene expression. ResultsVSMCs were readily transduced with different kinds of retroviral vectors. The bacterial neo r gene-transduced VSMCs were successfully selected with G418. The G418-selected VSMCs could express the transduced genes at a level comparable to NIH3T3. The level of transgene expression did not appear to be affected by the increasing number of passages. CONCLUSION: The results demonstrate an efficient transduction of VSMCs by retroviral vectors in vitro and an sustained expression of retrovirally transduced genes in VSMCs. VSMCs could be one of the ideal target cells for ex vivo cardiovascular gene therapy employing retroviral vector.
Animals ; Cats ; Gene Expression ; Genetic Therapy* ; Longevity ; Muscle, Smooth, Vascular* ; Retroviridae ; Rivers ; Transgenes ; Zidovudine

Genetically-engineered mouse (GEM) models have provided significant contributions to our understanding of cancer biology and developing anticancer therapeutic strategies. The development of GEM models that faithfully recapitulate histopathological and clinical features of human cancers is one of the most pressing needs to successfully conquer cancer. In particular, doxycycline-inducible transgenic mouse models allow us to regulate (induce or suppress) the expression of a specific gene of interest within a specific tissue in a temporal manner. Leveraging this mouse model system, we can determine whether the transgene expression is required for tumor maintenance, thereby validating the transgene product as a target for anticancer drug development (target validation study). In addition, there is always a risk of tumor recurrence with cancer therapy. By analyzing recurrent tumors derived from fully regressed tumors after turning off transgene expression in tumor-bearing mice, we can gain an insight into the molecular basis of how tumor cells escape from their dependence on the transgene (tumor recurrence study). Results from such studies will ultimately allow us to predict therapeutic responses in clinical settings and develop new therapeutic strategies against recurrent tumors. The aim of this review is to highlight the significance of doxycycline-inducible transgenic mouse models in studying target validation and tumor recurrence.
Animals ; Biology ; Doxycycline ; Drug Delivery Systems ; Humans ; Mice* ; Mice, Transgenic ; Recurrence ; Transgenes ; United Nations

Molecular nuclear cardiac imaging has included Tc-99m Annexin imaging to visualize myocardial apoptosis, but is now usually associated with gene therapy and cell-based therapy. Cardiac gene therapy was not successful so far but cardiac reporter gene imaging was made possible using HSV-TK (herpes simplex virus thymidine kinase) and F-18 FHBG (fluoro-hydroxymethylbutyl guanine) or I-124 FIAU (fluoro-deoxyiodo-arabino-furanosyluracil). Gene delivery was performed by needle injection with or without catheter guidance. TK expression did not last longer than 2 weeks in myocardium. Cell-based therapy of ischemic heart or failing heart looks promising, but biodistribution and differentiation of transplanted cells are not known. Reporter genes can be transfected to the stem/progenitor cells and cells containing these genes can be transplanted to the recipients using catheter-based purging or injection. Repeated imaging should be available and if promoter are varied to let express reporter transgenes, cellular (trans) differentiation can be studied. NIS (sodium iodide symporter) or D2R receptor genes are promising in this aspect.
Apoptosis ; Catheters ; Genes, Reporter ; Genetic Therapy ; Heart ; Linear Energy Transfer ; Molecular Imaging ; Myocardium ; Needles ; Thymidine ; Transgenes

Induced pluripotent stem (iPS) cells were originally generated from mouse fibroblasts by enforced expression of Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc). The technique was quickly reproduced with human fibroblasts or mesenchymal stem cells. Although having been showed therapeutic potential in animal models of sickle cell anemia and Parkinson's disease, iPS cells generated by viral methods do not suit all the clinical applications. Various non-viral methods have appeared in recent years for application of iPS cells in cell transplantation therapy. These methods mainly include DNA vector-based approaches, transfection of mRNA, and transduction of reprogramming proteins. This review summarized these non-viral methods and compare the advantages, disadvantages, efficiency, and safety of these methods.
Animals ; Cellular Reprogramming ; Humans ; Induced Pluripotent Stem Cells ; physiology ; Transduction, Genetic ; Transfection ; Transgenes

BACKGROUND: Adenovirus (Ad) vectors have been widely used for many gene therapy applications because of their high transduction ability and broad tropism. However, their utility for cancer gene therapy is limited by their poor transduction into cancer cells lacking the primary receptor, coxsackievirus and adenovirus receptor (CAR). METHODS: To achieve CAR-independent gene transfer via Ad, we pretreated Ad with 1,2-dioleoyl-3- trimethylammonium propane (DOTAP) and analyzed their transduction efficiency into cancer cells in vitro and in vivo comparing with the virus alone. RESULTS: Treatment of DOTAP significantly increased adenoviral gene transfer in tumor cells in vitro. Moreover, DOTAP at an optimum dose (10 microngram/ml) enhanced IL-12 transgene expression by fivefold in tumor, and twofold in serum after intratumoral injection of adenovirus expressing IL-12N220L (Ad/IL-12N220L). In addition, cotreatment of DOTAP decreased tumor growth rate in the Ad/IL-12N220L-transduced tumor model, finally leading to enhanced survival rate. CONCLUSION: Our results strongly suggest that DOTAP could be of great utility for improving adenovirus-mediated cancer gene therapy.
Adenoviridae* ; Genes, Neoplasm ; Genetic Therapy ; Interleukin-12 ; Liposomes ; Propane ; Survival Rate ; Transgenes ; Tropism

STUDY DESIGN: In vivo study to determine the immune effects to adenoviral vector encoding LMP-1 cDNA in rabbit. OBJECTIVE: To quantify the immune effect of Ad5-LMP-1 in the rabbit during the therapeutic gene transfer. SUMMARY OF LITERATURE REVIEW: One of the major limitations in the use of adenoviral vector for gene therapy is the immune response and it made the poor transduction efficiency when re-administrated. Adenoviral antigen plus those derived from transgene expression in transduced cell contribute to cellular, humoral and non-specific immune response constitutes barriers to successful gene therapy. Therefore, the animal immune model will be mandatory to study the immune impact. MATERIALS AND METHOD: We i.v. injected Ad5-betaGal to total 24adult NZW rabbits; 1x108, 1x109, 1x1010, 1x1011v.p. to each 6 rabbits allowed them to develop immune response. Six non-immunized animals were used as control. Adenovirus antibodies were measured at 0, 4, 8, 16, 20 weeks. Group I. 6 control rabbit underwent spinal arthrodesis at 4 weeks (n=3) and 16 weeks (n=3) with 4 million cells and MOI of 4. Group II. 6 rabbit underwent spinal arthrodesis at 4 weeks after injection of 108 p.f.u virus (n=3) and 16 weeks (n=3). Group III. six 109 immunized rabbits, Group IV. six 1010 immunized rabbits, Group V. six 1011 immunized rabbits, underwent spinal arthrodesis at 4 and 16 weeks after injection. Total anti-Ad Ig and neutralizing antibody titer was measured on the 0. 4. 8, 16, 20 weeks after injection. RESULTS: Group I. All 6 non-immunized rabbits had solid spine fusions at 4 and 16 weeks. Group II. All 3immunized rabbits had not spine fusions at 4 weeks and all three had solid spine fusion at 16 weeks. Group III. None of them (n=6) immunized rabbits had spine fusion at 4 and 16weeks, but some bone formation was observed at 16 weeks. Group IV, V. None of them immunized rabbits had bone formation. The anti-Ad5 Ig and neutralizing Ab were detected and peaked at the 4weeks and significantly dropped off 16 weeks after injection. CONCLUSION: This experiment revealed that a small dose of adenovirus elicited an enough immune response that inhibited the bone formation. Because majority of human posses the Ab against adenovirus, it will be mandatory to overcome immune response in adenoviral vector gene therapy.
Adenoviridae ; Animals* ; Antibodies ; Antibodies, Neutralizing ; Arthrodesis ; DNA, Complementary ; Genetic Therapy* ; Humans ; Models, Animal* ; Osteogenesis ; Rabbits ; Spinal Fusion* ; Spine ; Transgenes

OBJECTIVETo investigate the targeting expression of TRAIL gene driven by human telomerase reverse transcriptase (hTERT) promoter in SACC-83 cell with telomerase activity.

METHODSAdenovirus vector AdTERT-TRAIL was constructed by homologus recombination. After transfecting AdTERT-TRAIL into SACC-83 cell and HEL cell, its effect on these cells in vitro was investigated using RT-PCR technique, MTT method and flow cytometry.

RESULTSAfter transfection of AdTERT-TRAIL, expression of extrinsic TRAIL gene driven was detected in SACC-83, the proliferation of SACC-83 cell showed significant inhibitory effect (the relative cell viability was 49.70%) and its apoptotic rate was promoted (30.49%), whereas no TRAIL gene was detected in HEL cell, also no inhibitory effect was observed in HEL cell and its apoptotic rate showed little change.

CONCLUSIONAdenovirus vector AdTERT-TRAIL was successfully constructed, which can be used to induce expression of TRAIL gene in SACC-83 cell with targeting effect.

Carcinoma, Adenoid Cystic ; Cell Line ; Cell Line, Tumor ; Genetic Vectors ; Humans ; Promoter Regions, Genetic ; Telomerase ; Transfection ; Transgenes