Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-10-19
2001-08-07
Nguyen, Dave Trong (Department: 1633)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S320100, C435S455000, C435S458000, C424S093200
Reexamination Certificate
active
06271207
ABSTRACT:
BACKGROUND OF THE INVENTION
A. Field of the Invention
The present invention relates to the field of gene expression, generally, and more specifically it adduces the expression recombinant transgenes in host cells. The invention may be exploited in the production of recombinant proteins in vitro or in gene therapy in vivo.
B. Related Art
The ability to express foreign genes in host cells has become a pivotal tool in molecular biology. For example, expressing proteins in host cells in vitro can lead to the large scale production of the protein for use in research or therapy. Examples of proteins that could be used in this manner are hormones, such as insulin, or cytokines, such as the interleukins. Scientists are constantly seeldng ways to maximize expression of tansgenes when they are imported into host cells.
Another important technology affected by foreign gene expression is gene therapy. Attaining high level expression in specific target cells is a key aspect of gene therapy, too. Numerous parameters have been varied in an effort to achieve higher levels of expression including the mode of gene trasduction, the vector, the promoter, as well as the dose and routes of administration.
Recently Son & Huang (1994) reported that exposure of CDDP-resistant ovarian carcinoma cells to CDDP prior to liposome-mediated gene transfer resulted in enhanced gene transduction. This study utilized an ovarian cancer cell line, 2008, that rapidly acquire CDDP-resistance following exposure. The cells in this study were exposed to CDDP for four to six weeks prior to gene transfer (in vitro) or exposed once, one week prior to gene transfer (in vivo). According to the authors, their data only indicate that CCDP-resistant cells show improved gene transduction. Thus, from this study, it is unclear whether CDDP-sensitive cells would provide the same results. It also is unclear whether the effect was tied to liposomal transfection methods, or could be more broadly applied.
A similar phenomenon has been observed in primary human foreskin fibroblasts infected with adeno-associated virus following brief exposure to high concentrations of CDDP or other DNA-damaging agents (Alexander et al., 1994; Russell et al., 1995). In this system, these non-malignant cells were rendered more susceptible to transduction by sublethal, but relatively high levels of DNA damaging agents. The treatments were conducted for 16 to 20 hours, followed immediately by transduction. The cause of the increased expression was not correlated with other transduction methodologies. Moreover, it was unclear from the data whether the treatment rendered the cells more competent for AAV transduction, whether increased uptake of AAV was induced or whether increased expression of AAV occurred following internalization.
Thus, it is clear that the prior art does not provide a clear picture, with respect to the effect of DNA-damaging agents, of their effects on the transduction and expression of transgenes in various host cells. There remains a need for a better understanding of these phenomena and for increasing the expression of transgenes in transduced cells.
SUMMARY OF THE INVENTION
It is, therefore, an object of the present invention to provide improved methods for expression of transgenes in host cells. This applies both to the in vitro and in vivo applications and includes a variety of different gene delivery systems, DNA-damaging agents, transgenes and target cells. More specifically, the invention provides for the enhancement of gene expression by providing a DNA damaging agent, preferably to a dividing cell, prior to administration of a vector containing a gene or genes of interest
In one embodiment, the invention provides a method for enhancing the expression of a transgene comprising (a) contacting a target cell with a DNA-damaging agent; (b) removing the DNA-damaging agent from the target cell; and (c) transferring the transgene into the target cell between about 1-3 days after removing the DNA-damaging agent. More preferably, the tranfser of the gene occurs at about 2 days after removal. The target cell preferably is a dividing cell, and more preferably is a tumor cell.
The cell may be drug sensitive or drug insensitive.
The DNA-damaging agent is selected from the group consisting of cisplatin, carboplatin, VP16, teniposide, daunorubicin, doxorubicin, dactinomycin, mitomycin, plicamycin, bleomycin, procarbazine, nitrosourea, cyclophosphamide, bisulfan, melphalan, chlorambucil, ifosfamide, merchlorehtamine, and ionizing radiation.
Transfer of the transgene is accomplished by a technique selected from the group consisting of liposome-mediated transfection, receptor-mediated internalization and viral infection.
The transgene may be a tumor suppressor, such as p53. The transgene may be under the transcriptional control of a promoter, for example, the CMV IE promoter. Further, the trangene may have, in operable relation thereto, a polyadenylation signal, for example, the SV40 polyadenylation signal The transgene may be carried in an adenoviral vector.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
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Cristiano Richard J.
Nguyen Dao
Board of Regents , The University of Texas System
Fulbright & Jaworski
Nguyen Dave Trong
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