Adenovirus E4 proteins for inducing cell death

Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai

Reexamination Certificate

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C435S320100, C435S267000, C435S455000, C536S023100, C424S093200

Reexamination Certificate

active

06730662

ABSTRACT:

BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to a pharmaceutical agent(s) to induce cell death for use in treating conditions which involve inappropriate cell survival.
(b) Description of Prior Art
Replication of human adenoviruses in terminally differentiated epithelial cells requires an efficient mechanism to induce cellular DNA synthesis. This induction permits replication of viral DNA and production of progeny virus. Human adenoviruses infect and kill epithelial cells very efficiently. Cell death occurs by apoptosis and virus spread occurs through endocytosis by surrounding cells.
Products of early region 1A (E1A) of the adenovirus genome induce cell DNA synthesis and are largely responsible for cell transformation by adenoviruses. E1A produces two major mRNAs of 13S and 12S which encode proteins of 289 and 243 residues (289R and 243R, respectively) that are identical except for the lack in 243R of a central 46-amino acid sequence, termed conserved region 3 or CR3, as schematically depicted in FIG.
1
A. Two additional regions present in the common sequence encoded by exon 1 of both E1A mRNAs are also conserved in all human adenovirus serotypes and have been termed CR1 and CR2. E1A products induce DNA synthesis through ,complex formation between CR2 and CR1 and the retinoblastoma tumor suppressor pRB and related p107 and p130 proteins, or between the amino terminus and CR1 and the transcriptional modulator p300 and possibly related proteins (Corbeil, H. B. et al., 1994, J. Virol. 68: 6697-6709). E1A-289R also activates expression of the early viral transcription units E2, E3, and E4, and certain cellular genes at least in part through interactions with transcription factors and basal transcription machinery requiring CR3 (Teodoro, J. G. et al., 1995, Oncogene 11: 467-474). In addition to CR3, transactivation of the E4 promoter has also been shown to rely to some degree on two regions encoded by the second exon of 13S mRNA, termed auxiliary regions 1 and 2, or AR1 and AR2. Production of stably transformed cells requires early region 1B (E1B) which encodes polypeptides of 19 and 55 kDa that are individually capable of cooperating with E1A via separate but additive pathways (McLorie, W. et al., 1991, J. Gen. Virol. 72: 1467-1471).
Considerable evidence indicates that a major function of E1B proteins in lytic infection and cell transformation is to suppress cytotoxic effects and apoptosis induced by expression of E1A. Without E1B, the toxicity of E1A products results in the death of E1A-transformed cells and a reduction in the yield of progeny due to the early demise of productively infected cells. E1A proteins can cause apoptosis by a process mediated by the tumor suppressor p53, which controls growth arrest and programmed cell death pathways (Teodoro, J. G. et al., 1995, Oncogene 11: 467-474). Expression of E1A products results in the elevation of p53 levels. The 55 kDa E1B protein binds to p53 and blocks both p53-mediated activation of gene expression and apoptosis (Teodoro, J. G. et al., 1994, J. Virol. 68: 776-786). The 19 kDa E1B protein appears to suppress apoptosis by a mechanism that is functionally analogous to that of the cellular proto-oncogene product Bcl-2 (Nguyen, M. et al., 1994, J. Biol. Chem. 269: 16521-16524). Cells infected with adenovirus mutants which fail to express the 19 kDa protein display enhanced cytotoxicity and extensive degradation of both cellular and viral DNA into nucleosome sized fragments (McLorie, W. et al., supra; Teodoro, J. G. et al., 1995, Oncogene 11: 467-474). At later times, even in the presence of E1B proteins, infected cells suffer apoptotic death and viral progeny spread to neighboring cells through endocytosis of cell fragments. In addition to the induction of DNA synthesis and cell transformation, the large 289-residue (289R) E1A protein also transactivates expression of all early viral genes, including early regions 1A, 1B, 2, 3 and 4 (reviewed in Teodoro, J. G. et al., 1995, Oncogene 11: 467-474).
It would be highly desirable to be provided with a pharmaceutical agent for induction of apoptosis when such induction is useful in the treatment of human diseases which involve inappropriate cell survival.
SUMMARY OF THE INVENTION
In accordance with the present invention, we have used a genetic approach to identify the role of individual E4 proteins in the induction of p53-independent apoptosis. Our results indicate the E4 death proteins, E4orf4 or E4orf6, are responsible for induction of p53-apoptosis in transformed, but not untransformed, cells. Thus, E4orf4 and E4orf6 are both powerful inducers of p53-independent cell death. This discovery has significant ramifications for both apoptosis-inducing therapeutics and drug screens.
In a first aspect, the invention provides a method of increasing apoptosis in a cell by administering to the cell an apoptosis inducing amount of an E4orf6 polypeptide or an apoptotic fragment thereof. In a preferred embodiment of this aspect, the apoptosis is p53-independent.
In a second aspect, the invention provides a method of increasing apoptosis in a mammal which includes providing a transgene encoding an E4orf6 polypeptide or an apoptotic fragment thereof to a cell of the mammal. The transgene is positioned for expression in the cell, and preferably encodes E4orf6.
In a third aspect, the invention provides a method of increasing apoptosis in a cell which includes administering to the cell a compound which increases E4orf6 biological activity. In various preferred embodiments, the compound is E4orf6 mRNA, or increases the stability of E4orf6.
In a preferred embodiment of the first and third aspects of the invention, the cell is in a mammal, preferably a human.
In a fourth aspect, the invention provides a method of increasing apoptosis in a cell by administering to the cell an apoptosis inducing amount of an E4orf4 polypeptide or an apoptotic fragment thereof. In a preferred embodiment of this aspect, the apoptosis is p53-independent.
In a fifth aspect, the invention provides a method of increasing apoptosis in a mammal which includes providing a transgene encoding an E4orf4 polypeptide or an apoptotic fragment thereof to a cell of the mammal. The transgene is positioned for expression in the cell, and preferably encodes E4orf4.
In a sixth aspect, the invention provides a method of increasing apoptosis in a cell which includes administering to the cell a compound which increases E4orf4 biological activity. In various preferred embodiments, the compound is E4orf4 mRNA, or increases the stability of E4orf4.
In a preferred embodiment of the fourth and sixth aspects of the invention, the cell is in a mammal, preferably a human.
In a seventh aspect, the invention provides a method of increasing apoptosis in a cell by administering to the cell an apoptosis inducing amount of a composition which includes an E4orf6 polypeptide or an apoptotic fragment thereof and an E4orf4 polypeptide or an apoptotic fragment thereof. In a preferred embodiment of this aspect, the apoptosis is p53-independent.
In an eighth aspect, the invention provides a method of increasing apoptosis in a mammal which includes providing a first transgene encoding an E4orf6 polypeptide or fragment thereof and a second transgene encoding an E4orf4 polypeptide or fragment thereof to a cell of the mammal. The first and second transgenes are positioned for expression in the cell and, preferably, encode E4orf6 and E4orf6, respectively.
In a ninth aspect, the invention provides a method of increasing apoptosis in a cell which includes administering to the cell a composition which includes a first compound which increases E4orf6 biological activity and a second compound which increases E4orf4 biological activity. In various preferred embodiments, the first compound is E4orf6 mRNA, or increases stability of E4orf6, and the second compound is E4orf4 mRNA or increases stability of E4orf4.
In a preferred embodiment of the seventh and ninth aspects of the invention, the cell is in a mammal, preferably a human.
In preferred

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