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1

In vitro and in vivo study of the expression vector encoding vascular endothelial growth factor

100%
Malecki M., Przybyszewska M., Janik P.
Archivum Immunologiae et Therapiae Experimentalis
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2001
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vol. 49
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issue 3
243-246
EN
Vascular endothelial growth factor (VEGF) is an angiogenic cytokine with potential therapeutic applications in human diseases. It is a mitogen primarily for endothelial cells. The transfer of the cDNA encoding VEGF to ischemic tissues, which cannot be revascularized otherwise, represents a novel and promising approach to the treatment of vascular disorders. In this work the VEGF165 cDNA was cloned into the expression vector pSecTag2B. The activity of the construct was studied in cell culture as well as in vivo. Western blotting study showed that the cells transfected with the vector secreted significantly higher amounts of VEGF to the culture medium than the non-transfected cells. In vivo study revealed an increased number of new vessels in animals injected with vector encoding VEGF as compared with empty plasmid. Also, tumor cells transfected with the VEGF plasmid exhibited extensive vascularization.
2

AAV vectors for gene therapy

100%
Rutkowski A., Jazwa A., Jozkowicz A., Dulak J.
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2007
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issue 3
33-44
EN
This paper reviews the principles of the AAV vectors' generation. It describes various methods for their production and purification, as well as ways for increasing the efficiency and selectivity of the transduction by means of capsid modifications and use of various AAV serotypes. The second part of the article briefs clinical trials carried out so far with the use of the AAV vectors, particularly emphasizing the differences between feasibilities of vectors based on AAV and other virus types.
3

Gene therapy

100%
Jura J.
Biotechnologia
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1998
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issue 3
14-20
EN
Human gene therapy is one of the methods of the new millennium. There are over 100 protocols of gene therapy accepted by ethics committes and applied succesfully in clinics.
4

Production of adenoviral vectors of first generation ? optimization of method

80%
Stopa M., Jozwa A., Mleczko J., Dulak A., Jozkowicz A.
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2007
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issue 3
98-122
EN
The paper presents the production of adenoviral vectors (AdV) containing beta-galactosidase (Adbeta-gal), from the transfer of recombinant viral DNA into packing cell line (HEK293) to the titration of viral particles. Optimisation of the methods (preparation of DNA for transfection, adsorption time during the infection of cells, amount of serum in the medium, time-point of vector isolation) enables obtaining a titer of up to 1010 IU/mL. Adbeta-gal were titrated with several methods, with beta-gal in situ staining used as a reference. We found that the most suitable titration method of the vectors containing other than reporter genes was the Rapid Titer ELISA kit?.
5

Regulation of gene expression in plasmid vectors: doxocyclin-dependent and hypoxia-regulated systems

80%
Golda S., Kucharzewska P., Cisowski J., Florczyk U., Zagorska A., Jazwa A., Loboda A., Jozkowicz A., Dulak J.
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2007
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issue 3
82-97
EN
Regulation of gene expression in gene therapy is crucial for obtaining the therapeutic effects, thanks to limitation of transgene activity to the selected cells in a given time. In this paper we have focused on plasmid expression systems regulated by doxycycline or hypoxia. We have described in details the structure, regulatory elements and biological applications of 1) the modified, commercially available Tet-On system, expressing doxycycline-controlled b-galactosidase and, 2) hypoxia-activated FGF-4/VEGF expression plasmid containing the hypoxia responsive sequence. The presented expression systems can also be used in viral vectors, enabling not only regulated, but also high and long-term expression of transgenes.
6

The use of E.coli cytosine deaminase gene as an example of suicide gene therapy of cancer

80%
Missol E., Sochanik A., Szala S.
Biotechnologia
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1996
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issue 4
80-90
EN
Suicide genes encode enzymes, which convert nontoxic substrates to toxic products.These genes are proposed for selective killing of tumor cells in cancer gene therapy.In this review we demonstrate preliminary results of direct in vivo transfer of cytosine deaminase (CD) gene from E.coli into murine B16(F10) melanoma cells via cationic liposomes.
7

Construction of plasmid bicistronic vectors and their application for in vitro transfection

80%
Kucharzewska F., Zagorska A., Leja J., Jazwa A., Gozdecka M., Jozkowicz A., Dulak J.
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2007
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issue 3
66-81
EN
Internal ribosome entry site (IRES) sequences, which stand for the basic element of cap 5'-independent translation, are currently widely used to coexpress heterologous genes from one plasmid. In this study construction of four bicistronic plasmids containing IRES and application of these vectors for transfection of in vitro cultured cells were described. The obtained data show that constructed bicistronic plasmids are very efficient in vitro in terms of simultaneous expression of fibroblast growth factor-4 (FGF-4) and vascular endothelial growth factor (VEGF) or one of these factors and green fluorescent protein (GFP) from one plasmid. Interestingly, expression of two genes, although simultaneous, is not equal. It has turned out that IRES-dependent mRNA translation is less efficient than cap 5'-dependent translation of the first gene, which should be taken into account during construction of bicistronic plasmids.
8
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Treatment of lysosomal storage disorders: Focus on the neuronal ceroid-lipofuscinoses

80%
Pierret C., Morrison J. A., Kirk M. D.
|
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issue 3
429-442
EN
Recent advances in our understanding of lysosomal storage disorders (LSDs) may lead to new therapies to treat the neuronal ceroid-lipofuscinoses (NCLs). In this review, enzyme replacement therapy, gene therapy, cell-mediated therapy and pharmaceutical treatments are considered across the LSDs and extended to therapies for the NCLs. It is likely that a combination of approaches will produce the most beneficial clinical outcome for treatment of pathologies displayed by the NCLs.
9

Optimization of transduction of chosen cell lines using adenoviral vetors of the first generation

80%
Stopa M., Dulak A., Jozkowicz A.
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2007
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issue 3
123-140
EN
Efficacy of adenoviral vectors (AdV) was checked by transduction of six different cell lines (COS-7, HUVEC, HMEC-1, NIH 3T3, HaCaT, and B16(F10)) with the vectors containing reporter gene beta-galactosidase (Adbeta-gal). We optimised the adsorption time and dose of Ad beta-gal. The transduction with efficacy of up to 100% was obtained for the doses 10-100 IU/cell. In order to examine the effect of transduction procedure on biology of the cells, we measured the production of major proinflammatory cytokines. In several cell lines (HMEC-1, HUVEC, HaCaT), we found the induction of IL-6 synthesis and decrease in the cell viability, particularly in the case of the highest Adbeta-gal dose. However, the treatment with adenoviral vectors did not induce TNF generation and did not modify proliferation of cells.
10

Gene therapy by means of liposomes

80%
Kunio Y.
Biotechnologia
|
1996
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issue 4
17-25
EN
One great advantage of liposome-encapsulated genes for gene theraphy is its safeness.But a shortcoming is its low transfection effeciency.To overcome this shortage, we devised cationic large uniamellar vesicles for efficient and transfer of the gene for its expression.Recently, we devised a simple method to prepare cationic multilamellar large vesicles.By use of these vesicles, malignant glioma in an experimental model was successfully cured.
11

DNA viruses and genetic modification of dendritic cells

80%
Jenne L., Steinkasserer A.
|
|
issue 4
273-279
EN
An alternative approach to the use of tumor-peptide-loaded dendritic cells (DC) in immunotherapy would be the use of genetically modified DC using viral vectors expressing tumor-associated antigens (TAA). However, viruses have developed several immune escape mechanisms and, thus, one has to study the interaction between viruses and DC before these viruses can be used as an alternative strategy. Here we report that vaccinia virus (VV) as well as herpes simplex virus type-1 (HSV-1) are able to potently infect monocyte-derived DC, however, this infection leads to the inhibition of the DC-mediated T cell stimulation in vitro.
12

Adeno-associated virus vectors in gene therapy

80%
Kamieniarz K., Gozdzicka-Jozefiak A.
Biotechnologia
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2005
|
issue 1
79-94
EN
The development of targeted vectors, capable of tissue-specific transduction, remains one of the most important aims of vector modification for gene therapy applications. The gaining popularity of recombinant vectors based on adeno-associated viruses (rAAV) in gene therapy can be attributed to their lack of pathogenicity, added safety due to their replication defectiveness, relatively low immunogenicity and their ability to mediate long-term expression in a variety of tissues. The major shortcoming of these vectors is their small packaging capacity. AAV vectors have already broad utility in the therapy of many diseases, including neurological disorders and various types of cancer. Moreover, they can also serve as transfer vehicles for DNA vaccines.
13

New strategies for application of plasmid and viral vectors in gene therapy

80%
Jozkowicz A., Dulak J.
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2007
|
issue 3
7-21
EN
The success of gene therapy depends on development of efficient and safe delivery vectors. Recently, significant progress has been achieved in application of various non-viral delivery methods and numerous modified viral vectors for experimental gene therapy. Some of those vectors have been also successfully used in clinical trials of gene therapy. Further progress in this still promising, although not-fulfilling all expectations, treatment is dependent on the careful evaluation of the modes of transgene delivery and regulation of transgene expression in the diseased tissues. In this review the current progress in development of some of the tools for therapeutic gene transfer is discussed.
14

Gene therapy of cardiovascular diseases

80%
Dulak J.
|
EN
Gene therapy is suggested to be beneficial for the treatment of diseases which are difficult or impossible to be cured by classical pharmacotherapy. Thus, the transfer of genetic material of potential therapeutic properties may be particularly helpful in the treatment of hereditary genetic disorders and cancer. However, serious technical obstacles, especially the lack of efficient vectors for the delivery of therapeutic genes have so far prevented the achievement of convincing therapeutic effects. On the other hand, the complications of atherosclerosis, such as heart or peripheral ischaemia, seem to be good candidates for genetic strategies aimed particularly at stimulation of angiogenesis. As in this case therapeutic genes should be expressed locally and in a short time there is a good chance that the application of this therapy will take place earlier than in the case of other disorders. In this paper the first gene therapy clinical trials in human cardiovascular diseases are described. The latest investigations on the basic mechanisms of the blood vessel wall physiology and relationships between factors involved in angiogenesis are also briefly mentioned.
15

Methods for the titration of AAV vectors

80%
Rutkowski A., Jazwa A., Popowa S., Jozkowicz A., Dulak J.
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2007
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issue 3
157-166
EN
Adeno-associated viral (AAV) vectors are promising tools for gene therapy. However, for trustworthy comparison of the results produced from different clinical trials, the exact amount of the used infectious vector particles must be known. We have produced AAV using a commercially available system and compared three common methods for the quantification of the number of produced vectors: ELISA, dot-blot and quantitavive PCR (qPCR). Although ELISA is a very reproducible and precise method, it is able only to determine the number of viral capsids in the vector preparation, also those which contain no genetic material and are therefore useless. With this method we established that we are able to produce ~ 6.5 x 1011 viral capsids/mL. Dot-blot assay determines the number of genomic particles in the vector preparation in a quite precise manner, but it is a very labor- and time-consuming method. qPCR is also a method determining the number of genomic particles. It is, however, much faster and simpler than the dot-blot assay. Both dot-blot and qPCR gave similar results (~ 4 x 1011 viral genomes/mL), which indicated only about 2/3 of the produced vectors containing genetic material. Our results show that qPCR is the most convenient and reliable method for quantification of AAV vectors and we believe it could be routinely used to titer the vectors prior to their usage in clinical trials.
16

Optimization of methods for the production of AAV vectors

80%
Jazwa A., Rutkowski A., Golda S., Rehhahn A., Jozkowicz A., Dulak J.
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2007
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issue 3
141-156
EN
One of the conditions of effective gene therapy is the choice of a proper gene carrier that will efficiently deliver the genetic material to the damaged tissue without causing deleterious side-effects. Adeno-associated viral vectors (AAV) have emerged as attractive tools for gene therapy, because of their broad tissue tropism, long-term transgene expression, and lack of human pathology. Nevertheless, difficulty in preparing and purifying this viral vector in large quantities remains a major obstacle for evaluating AAV vectors in clinical trials. In this article, we compare different methods for AAV production in order to optimize the conditions of AAV preparation to the scale and purity required for clinical and potential commercial applications.
17

The most important features of adenoviral vectors

80%
Stopa M., Dulak J., Jozkowicz A.
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2007
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issue 3
22-32
EN
One of the major hurdles to successful gene therapy is the ability to efficiently introduce a foreign nucleic acid into the tissue of interest. As adenoviruses posses ability to enter rapidly a mammalian host cell and achieve propagation, they are widely used as a tool for delivery transgenes. Recombinant adenoviral vectors of first generation (AdV) contain an expression cassette with exogenous genes and are made replication deficient by the deletion of the E1/E3 region. They offer many advantages for gene delivery: ability to transduce a wide variety of cell types in a cell-cycle independent manner, easy and cheap propagation process to high titers and low pathogenicity for humans. However, AdV also have some disadvantages, namely cytotoxity, immunogenity, transient expression of transgene, which are mostly important in case of clinical trials. Despite those limitations and development of more sophisticated adenoviral systems (helper-dependent adenoviral vectors), AdV of first generation are still widely used for trasnducting different cell types in vitro, especially those that are refractory to other gene transfer methods, as well as in gene therapy clinical trials.
18

Gene therapy of cancer

71%
Gorny A., Wiznerowicz M., Mackiewicz A.
Biotechnologia
|
1995
|
issue 1
74-90
EN
Progress in genetic engineering has to lead to the development of efficient methods of transfer and expression of genes in eucariotic cells.This technology has been employed to kill cancer cells.Five major strategies of cancer gene therapy have been proposed and are currently verified in clinical trials.They include: (1) genetic cellular vaccines, (2) siuicide genes, (3) multidrug resistance genes, (4) HLA genes tansfer into cancer cells, and (5) repair of defective suppresor genes and/or removal of activated oncogene.
19

From neoplastic neural development to gene therapy of brain tumors ? IGF-I antisense and triple helix approaches

71%
Trojan J.
Biotechnologia
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2003
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issue 2
280-289
EN
IGF-I, insulin ? like growth factor I, seems to play a major role in the normal and tumoral development of the nervous system. Glioblastoma is the most frequent brain tumor in man and is usually fatal. Both human and rat glioma cells express high amounts of IGF-I. When rat glioma cells are transfected with vectors expressing either IGF-I antisense RNA or inducing IGF RNA ? DNA triple helix, the synthesis of IGF-I was stopped on translation or transcription levels, respectively. Down-regulation in the expression of IGF-I coincides with the reappearance of B-7 and MHC class I antigens at the surface of transfected cells. When injected subcutaneously, the transfected cancer cells initiate an immune reaction involving CD8+ lymphocytes, followed by tumor regression. The ?anti-gene? strategy for clinical therapy of glioblastoma, and other tumors expressing IGF-I such hepatomas were introduced in University Hospitals of Cleveland (USA), Shanghai ( China) , Krakow and Bydgoszcz (Poland).
20

The dog genome map and its use in mammalian comparative genomics

61%
Switonski M., Szczerbal I., Nowacka J.
Journal of Applied Genetics
|
2004
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vol. 45
|
issue 2
195-214
EN
The dog genome organization was extensively studied in the last ten years. The most important achievements are the well-developed marker genome maps, including over 3200 marker loci, and a survey of the DNA genome sequence. This knowledge, along with the most advanced map of the human genome, turned out to be very useful in comparative genomic studies. On the one hand, it has promoted the development of marker genome maps of other species of the family Canidae (red fox, arctic fox, Chinese raccoon dog) as well as studies on the evolution of their karyotype. But the most important approach is the comparative analysis of human and canine hereditary diseases. At present, causative gene mutations are known for 30 canine hereditary diseases. A majority of them have human counterparts with similar clinical and molecular features. Studies on identification of genes having a major impact on some multifactorial diseases (hip dysplasia, epilepsy) and cancers (multifocal renal cystadenocarcinoma and nodular dermatofibrosis) are advanced. Very promising are the results of gene therapy for certain canine monogenic diseases (haemophilia, hereditary retinal dystrophy, mucopolysaccharidosis), which have human equivalents. The above-mentioned examples prove a very important model role of the dog in studies of human genetic diseases. On the other hand, the identification of gene mutations responsible for hereditary diseases has a substantial impact on breeding strategy in the dog.
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