Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-11-24
2001-02-13
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
Reexamination Certificate
active
06187588
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to the field of genetic engineering, and particularly to the field of transfection of cells. More particularly, this invention provides a method for increasing the efficiency of transfection of cells by inhibiting cell death.
2. Description of Related Art
Transfection
Transfection of cells is routinely used as a genetic engineering tool. Transfection is the introduction of a foreign gene(s) into a target cell. Transfection methods can be useful in the areas of medicine, agriculture, pharmaceuticals, and Biomedical research. A particularly important application of transfection is gene therapy, wherein a foreign gene can be stably incorporated into a patient's genome thereby conferring upon the transfected cell, the ability to produce the product of the transfected gene.
The broad applicability of transfecting nucleic acid molecules into target cells, has led to the development of a number of protocols for performing transfections, many of which involve the use of carrier such as DEAE-dextran or calcium phosphate promoting the uptake of exogenous nucleic acids by cells. Other methods use cationic lipids for liposome induced uptake of nucleic acid sequences. Still other methods involve osmotic shock of cells or high-voltage electric pulses (electroporation) to create pores in cell membranes to facilitate uptake of nucleic acid molecules.
Electroporation is one of the common methods for transfection (Zimmermann, 1986,
Reviews of Physiology Biochemistry and Pharmacology.,
105:176-256; Hui, 1995,
Methods in Molecular Biology,
48:29-40). The high rate of transfection, simple control, and absence of biological contamination make electroporation a potentially promising method for gene therapy, especially in ex vivo cases (Bergan et al., 1996,
Blood
88:731-741). However, the electrotransfection efficiency for certain cell types, particularly normal or malignant lymphocytes, is low. The reasons for the poor results are not known. Thus, identifying the reasons for the low transfection efficiency of lymphoid cells could have a significant impact on gene therapy and human health.
Apoptosis
Apoptosis, or programmed cell death, plays a critical role in the development and differentiation of multicellular organisms, cell homeostasis, immune function, and aging (Kerr et al., 1972,
Br. J. Cancer
26:239-257; Sarin et al., 1996,
J. of Experimental Medicine
184:2445-2450; Vaux et al., 1996,
Proc. Natl. Acad. Sci. USA
93:2239-2244). Malfunctions of apoptosis are closely correlated with multiple forms of pathogenesis, including drug resistance and carcinogenesis (Vaux et al., 1998,
Nature
335:440-442; Miyashita et al., 1993,
Blood
81:151-159. Programmed cell death is an autonomous cellular process that is dependent upon the balance of relative levels of expression of members of the bcl-2 gene family (Oltvai et al., 1993,
Cell
74:609-619; Chittenden et al., 1995,
Nature
374:733-736). Apoptosis has been known to be responsible for death of traumatized cells. Previous studies demonstrated that transfection of cytokine-induced killer (CIK) T lymphocytes by receptor-mediated or cationic liposome-mediated transfection resulted in apoptosis (Ebert et al., 1997, 1997,
Gene Therapy,
4:296-302). It was observed that these transfection methods induced the secretion of high levels of TNF-&agr; by the transfected CIK T cells, which then activated the apoptotic pathway. Interestingly, only low levels of TNF-&agr; secretion were detected from CIK T cells transfected by either electroporation or retroviral gene transfer.
With efforts continuing toward understanding the role of cell death in transfection, there is an ongoing need for improving the efficiency of transfection so as to achieve better results with gene therapy.
SUMMARY OF THE INVENTION
The present invention provides a method for improving the efficiency of transfection. The method is based on the unexpected finding that DNA-uptake induces apoptosis, and comprises the steps of exposing the transfected cells to inhibitors of apoptosis. The cells may be exposed to the apoptosis inhibitors during and/or after transfection.
REFERENCES:
patent: 5563039 (1996-10-01), Goeddel et al.
Uhlmann et al., J. Biol. Chem. 273: 17926-17932, A potent cell death activity associated with transient high level expression of BCL-2, Jul. 1998.
Bird et al., Mol. Cell. Biol. 18: 6387-6398, Selective regulation of apoptosis: the cytotoxice lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without preturbing the Fas cell death pathway, Nov. 1998.
Tatsuta et al., J. Immunology 157: 3949-3957, Intracellular IL-1beta is an inhibitor of Fas-mediated apoptosis, 1996.
Hui Sek W.
Li Lin H.
Murphy Shawn P.
Sen Arindam
Health Research , Inc.
Hodgson Russ Andrews Woods & Goodyear LLP
Ousley Andrea
Schwartzman Robert A.
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