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1

AAV vectors for gene therapy

100%
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.
2

Therapeutic potential of heme oxygenase-1 in cardiovascular disease

88%
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.
3

Methods for the titration of AAV vectors

88%
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.
4

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

76%
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.
5

Optimization of methods for the production of AAV vectors

76%
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.
6

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

64%
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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