[Identifying strategies to clone viral genes into yeast expression vector]
The therapy of human diseases exploiting the powerful tools provided by modern cellular and molecular biology and gene technology is one of the great intellectual and technical challenges of present biology and molecular medicine. It is expected that gene and cell therapy can be used for the treatment of many and very common human diseases where conventional medical procedures fail to be successful.
While such therapeutic strategies are theoretically possible, their application is still limited by technical problems, among these are the delivery of transgenes into cells and tissues and the lack of efficient and safe vector systems. The final goal of all such strategies is to achieve the authentic expression of transgenes or nucleic acid sequences of therapeutic value in a specific cell type in the absence of undesired interactions with the host's genome and without the risk of cellular transformation or stimulating the host's immune system. This is rarely achieved using the conventional vector systems that are currently used for gene therapy.
It is apparently possible to transfect muscle cells very efficiently with naked DNA. Using this technique long term expression of a transgene has been observed, although the status of transferred DNA in the cell is yet poorly analyzed. Transfection of other cell types can be very inefficient and therefore appropriate vector systems have to be chosen. Unfortunately, currently used vectors for eukaryotic cells suffer from a number of limitations which makes them only partially useful for gene therapy of human diseases (Prince, 1998).
Integration into the host genome by for example retroviral vectors can lead to insertional mutagenesis and in all cases profound differences in the pattern and level of transgene expression have been observed. Recently, direct experimental evidence was provided that retroviral vectors can induce leukemia in mice and humans demonstrating the problems related with such integrating and, in particular, retrovirus-derived vectors ( [Li et al., 2002a], [Li et al., 2002b], [Marshall, 2000a], [Marshall, 2000b] and [Check, 2002]). On the other hand, transient expression as achieved for example with adenoviral vectors means that repeated transfections are required which can cause undesired immunological side effects in the host organism. For these reasons multiple transfections are sometimes not possible and in most cases not desirable. A solution to these problems could be the construction of vectors based on chromosomal regulatory elements which either behave in a way that they faithfully mimic gene expression from the natural context following integration or of vectors that are stably maintained and replicated as an episome in the target cell.
Gene expression in eukaryotic cells is regulated at many different levels. While DNA-binding proteins and their interaction with the basic synthetic machinery drives transcription, it is now clear that the efficiency and the precision of this process is strongly influenced by higher nuclear organization ( [Cook, 1999], [Cremer et al., 2000], [Cremer and Cremer, 2001] and [Jackson, 2002]). Nevertheless, it is generally believed that it is a limited number of DNA elements that is involved ...