Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-02-14
2001-09-04
Hauda, Karen M. (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C530S350000, C424S093100, C424S093200, C435S320100, C435S455000
Reexamination Certificate
active
06284880
ABSTRACT:
The present invention relates to a method for treating higher eukaryotic cells, particularly for introducing foreign material such as DNA into the cells.
There is a need for an efficient system for introducing foreign material, particularly nucleic acid, into living cells especially in the field of gene therapy. This involves locking genes into cells in order to synthesise therapeutically active gene products in vivo.
Standard methods for the transfection of cells make use of, inter alia, calcium phosphate (for use in vitro), cationic lipids (Felgner et al., 1993) or liposomes.
The technologies which are currently most advanced in the field of gene therapy make use of recombinant vector systems (retroviruses or adenoviruses) for transferring genes into the cell (Wilson et al., 1990; Kasid et al., 1990, WO 93/03769).
Alternative strategies for gene transfer are based on mechanisms which the cell uses for transporting macromolecules. One example of this is the introduction of genes into the cell by the route of receptor-mediated endocytosis (e.g. Wu and Wu, 1987, Wagner et al., 1990, and EP-A1 0388 758).
Studies of gene therapy in general and non-viral gene transfer methods in particular are provided by Mulligan, 1993, and by Cotten and Wagner, 1993.
For gene transfer with DNA/polycation complexes by means of receptor-mediated endocytosis, an improvement has been proposed which envisages using components on the basis of their ability to release the contents of endosomes, e.g. adenoviruses or fusogenic peptides. The use of the endosomolytic components brings about an increase in the efficiency of gene transfer by avoiding the breakdown of the DNA complexes internalised in the cell in the lysosomes (Curiel et al., 1991; Curiel et al., 1992a; Zatloukal et al., 1992; Cotten et al., 1992; Wagner et al., 1992; Curiel et al., 1992b; WO 93/07283). It has been proposed, among other things, to modify the adenoviruses by binding to polylysine. The adenovirus-polylysine conjugates can be complexed with DNA, together with conjugates of transferrin-polylysine, to obtain ternary transferrin-polylysine/adenovirus-polylysine/DNA complexes (Wagner et al., 1992). The complexes bind to transferrin and adenovirus receptors on the target cells. After the endocytosis the adenovirus causes the endosomes to open up, resulting in the release of the material from the endosome into the cytoplasm. This technique, which is also known as “transferrinfection” is more reliable than conventional viral techniques (Cotten et al., 1992).
Admittedly, transiently high expression values have been achieved with adenovirus-aided gene transfer based on receptor-mediated endocytosis, but because of toxic effects in some types of cell it has not been possible to maintain expression over a longer period of time. These toxic effects can be traced back to various defence responses of the host cell, shortly after the virus enters the cell. The mechanisms which regulate the activation of these responses have not yet been explained. Investigations with replication-deficient adenovirus mutants lead one to suppose that the responses of the host occur very early on, possibly even before the expression of the virus gene.
One component of this host response is activation of the “Interferon Responsive Genes” (genes which respond to interferon), most probably by activation of ISGF3, a transcription factor which responds to interferon, the binding sites of which are found upstream of a series of genes responding to interferon. One of the activated genes is the protein kinase p68, which is activated by double-stranded RNA. p68 is synthesised in an inactive form and, in the presence of dsRNA is subject to autocatalytic phosphorylation which activates the kinase, leading to phosphorylation and inactivation of the translation initiation factor eIF2a, thereby blocking the initiation of protein translation. It has also been reported that NF-
K
B is activated by dsRNA (Visvanathan and Goodbourn, 1989), which indicates that perhaps p68 directly phosphorylates I
K
B and activates NF-
K
B.
The type C-adenoviruses have two powerful mechanisms for preventing this stoppage of translation by the host:
The E1A gene products can directly disrupt the activation of ISGF3 (Gutch and Reich, 1991; Ackrill et al., 1991).
A second, again directly acting mechanism makes use of the VA1-genes. The gene product forms a stable secondary structure which can bind to the p68 kinase without activating the kinase, and which can prevent binding and activation by the actual activators (Manche et al., 1992; Mathews and Shenk, 1991).
Another inflammatory reaction mechanism as a result of the entry of a virus would consist in the activation of the inflammatory transcription factor NF-IL-6 and the secretion of the inflammatory cytokine IL-6 (Sehgal et al., 1988; Kishimoto et al., 1992). This factor, often in collaboration with NF-
K
B, in turn activates the IL-6-gene itself as well as a number of inflammatory response genes such as TNF and IL-1. It has recently been reported that IL-6 can in turn activate the transcription factor IRF 1 (“Interferon Response Factor 1”) which responds to interferon (Harroch et al., 1994). This would either be responsible for the interferon response to the entry of adenovirus or would intensify the response. Since the majority of the early genes of adenovirus type C contain binding sites for NF-IL-6, it may be assumed that activation of NF-IL-6 by adenovirus ensures that the virus gene expression cascade is triggered.
Another line of defence of the host cell would be the apoptotic response. It has long been known that mutations which map in the E1B 19K region are responsible for the deg-phenotype, an intensified cytopathic effect which is accompanied by the breakdown of the chromosomal DNA of the host (D'Halluin et al., 1979; Ezoe et al., 1981; Lai Fatt and Mak, 1982; Pilder et al., 1984; Subramanian et al., 1984; Takemori et al., 1984; White et al., 1984). Recent research has identified apoptosis which is caused by expression of an E1A growth signal in the absence of E1B (White and Stillman, 1987; White et al., 1994; Rao et al., 1992); the release of the transcription factor E2F from the Rb-protein, triggered by E1A, would appear to be involved in this apoptosis (Wu and Levine, 1994). It has been shown that the E1B 19K protein can function as a highly effective analogue of the host gene Bcl-2 which blocks the apoptosis (Rao et al., 1992; Debbas and White, 1993). The expression of each of these proteins may block an apoptosis triggered by various signals. In conjunction with the investigations with Myc, it has been found that growth signals determine a cell either for proliferation, if the appropriate reinforcing signals, e.g. Ras or Src, are available, or for apoptosis if only the Myc signals are available (Evan et al., 1992). A similar model for transformation induced by adenovirus E1 is supported by experiments which show that the apoptosis is triggered by E1A expression in the absence of E1B 19K (White and Stillman, 1987; Rao et al., 1992; Debbas and White, 1993). A recent investigation into the sindbis virus infection has shown that the expression of Bcl-2 in the host cell may be a critical factor in the outcome of a viral infection (Levine et al., 1993): in the absence of Bcl-2 the cell carries out apoptosis, thereby restricting the virus production to a short acute phase. In the presence of Bcl-2 expression, chronic virus production occurs because the apoptotic response to the acute phase infection is not achieved. Anti-apoptotic activities have also been identified in the Epstein-Barr virus (the BHER1 gene; Pearson et al., 1987), in the baculovirus (the p35 gene; Sugimoto et al., 1994) and in the African swine fever virus (the LMW5-HL gene; Neilan et al., 1993). This indicates that the apoptotic response to the virus infection occurs in many cases and viruses have developed strategies for blocking this response.
The experiments carried out with inactivated adenovirus capsids for gene transfer by means of receptor-mediated endocyto
Baker Adam
Chiocca Susanna
Cotten Matthew
Baker Anne-Marie
Boehringer Ingelheim International GmbH
Hauda Karen M.
Sterne Kessler Goldstein & Fox PLLC
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