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1

Gene therapy of cardiovascular diseases

100%
Dulak J., Jozkowicz A.
Biotechnologia
|
2004
|
issue 3
35-54
EN
Despite progress in pharmacotherapy, there is still a lack of efficient treatment of advanced stages of cardiovascular diseases. Therefore, great expectations are connected with gene therapy. First clinical experiments of gene transfer of vascular endothelial growth factor (VEGF) were carried out in 1994-1998 on patients with critical leg ischemia. They resulted in the improvement of vascularisation and prevented the amputation of the limbs. Promising results have been also obtained with DNA decoys binding the EF2 transcription factor. In this way, the expression of genes enhancing the proliferation of vascular smooth muscle cells have been inhibited and in consequence the narrowing of the lumen of arterio-venous bypasses have been inhibited. Moreover, the experimental data from animal models of cardiovascular diseases suggest that the improvement patients' conditions of can be obtained by the transfer of genes modulating the inflammatory processes which are the main underlying cause of atherosclerosis. Thus, the enhanced expression of heme oxygenase-1, nitric oxide synthases or superoxide dismutases may beneficially influence the functions of the vessels. This protective activity may involve moderate enhancement of the synthesis of VEGF. Owing to this, the improvement of angiogenesis can be obtained without the risk of side effects associated with very high, unregulated expression of this growth factor. Further progress in gene therapy of cardiovascular diseases will depend on the results of clinical trials, investigations of the mechanisms of disturbance in gene expression associated with cardiovascular diseases, as well as on the development of efficient methods of targeted delivery and regulation of the expression of therapeutic genes.
2

Nitric oxide production by cells infiltrating amphibian skin grafts. j

100%
Jozkowicz A., Plytycz B.
Folia Biologica
|
1997
|
vol. 45
73-78
EN
In vivo injection of the edible frog Rana esculenta with NOS inhibitor, L-NMMA caused prolongation of skin allograft and xenograft viability, statistically significant only in the latter case. In the present studies skin allo- and xenografts at the latent or rejection phase were excised from the hosts (Bufo bufo, R. temporaria, and R. esculenta) and incubated in vitro for 24 hrs in a medium only or in the presence of competitive (L-NMMA, L-NAME, L-aminoguanidyne) and noncompetitive (dexamethasone and cycloheximide) inhibitors of NO synthesis. In some experiments graft infiltrating cells were washed out and cultured separately from the respective skin fragments. The nitrite level was measured in the culture supernatant using Griess reagent. The nitrite level was negligible in the control skins, autografts, and xenografts depleted of graft infiltrating cells, as well as in allo- and xenografts excised at the rejection phase. In the case of grafts excised at the latent phase, the nitrite amount was substantial in supernatant from allografts and significantly higher in xenografts. A high level of nitrite was also present in supernatants from graft infiltrating cells. It is concluded that the NO contributes to some stages of the rejection process of the anuran skin grafts, this contribution being especially significant in the case of xenografts. The main source of this agent are graft infiltrating phagocytic cells.
3

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

100%
Jozkowicz A., Dulak J.
|
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.
4

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

81%
Stopa M., Dulak A., Jozkowicz A.
|
2007
|
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.
5

AAV vectors for gene therapy

81%
Rutkowski A., Jazwa A., Jozkowicz A., Dulak J.
|
2007
|
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.
6

The most important features of adenoviral vectors

81%
Stopa M., Dulak J., Jozkowicz A.
|
2007
|
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.
7

Production of adenoviral vectors of first generation ? optimization of method

71%
Stopa M., Jozwa A., Mleczko J., Dulak A., Jozkowicz A.
|
2007
|
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?.
8

Therapeutic potential of heme oxygenase-1 in cardiovascular disease

71%
Jazwa A., Florczyk U., Stepniewski J., Jozkowicz A., Dulak J.
Biotechnologia
|
2011
|
issue 2
166-179
EN
Heme oxygenase-1 (HO1) degrades heme to carbon monoxide (CO), biliverdin, and ferrous iron. Through these products, HO1 mitigates cellular injury by exerting anti-oxidant, anti-apoptotic, and anti-inflammatory effects. Several lines of evidence indicate that angiogenic factors, such as vascular endothelial growth factor A (VEGF) and stromal cell-derived factor 1 (SDF1), mediate their proangiogenic action in endothelial cells and endothelial progenitor cells through induction of HO1, and reciprocally, VEGF and SDF1 are enhanced by HO1 overexpression. Ferrous iron released during the breakdown of free heme by HO1 is an extremely pro-oxidative molecule that can be rapidly removed by ferritin. Of note, this iron sequestering protein also has been shown to exert some proangiogenic effects. Moreover, our recent data indicate that HO1 is an important mediator of differentiation and function of stem cells, including endothelial and myoblasts progenitors. All of this makes HO1 a promising target for novel cardiovascular therapies. The aim of this review is to discuss the existing knowledge and to propose the therapeutic approaches, which have to consider the necessity of tight regulation of HO1 expression.
9

Methods for the titration of AAV vectors

71%
Rutkowski A., Jazwa A., Popowa S., Jozkowicz A., Dulak J.
|
2007
|
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.
10

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

62%
Kucharzewska F., Zagorska A., Leja J., Jazwa A., Gozdecka M., Jozkowicz A., Dulak J.
|
2007
|
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.
11

Optimization of methods for the production of AAV vectors

62%
Jazwa A., Rutkowski A., Golda S., Rehhahn A., Jozkowicz A., Dulak J.
|
2007
|
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.
12

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

52%
Golda S., Kucharzewska P., Cisowski J., Florczyk U., Zagorska A., Jazwa A., Loboda A., Jozkowicz A., Dulak J.
|
2007
|
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.
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