Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1998-06-02
2003-12-16
Wilson, Michael (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S093100, C514S04400A
Reexamination Certificate
active
06663856
ABSTRACT:
BACKGROUND
0
F THE INVENTION
Cancer is a genetic disease that results from multiple genomic changes. These changes ultimately lead to the malfunction of cell cycle machinery and finally to autonomous cell proliferation. Neoplastic transformation involves four types of genes: oncogenes, tumor-suppressor genes, mutator genes, and apoptotic genes. Different types of cancer can involve alteration of any one or any combination of these genes.
In order for genomic alterations to lead to effects on cell proliferation, they must in some way affect the normal processes that control the cell cycle. The cell then becomes an autonomous unit capable of replicating without reliance on the external environment that normally signals a cell when to divide, to arrest, to differentiate, or to apoptose. Therefore, neoplastic development is ultimately a disturbance in either the positive regulators of the cell cycle (proto-oncogenes), the negative regulators (tumor suppressor genes), and/or apoptotic regulators of cell growth and proliferation. To develop more effective strategies for identification and treatment of various forms of cancer the mechanisms that operate within the cell cycle and how they might interlink, become disturbed, and be regulated must be considered.
Major advances in molecular biology, biochemistry and tumor biology have changed the way research scientists and clinicians conceptualize the management and treatment of cancer. Proteins that mediate cell cycle control are now being elucidated (e.g., p53 nuclear protein), while DNA technology provides ready access to the genes that control the events. Direct targeting of these proteins for either delivery or modification by conventional pharmacologic agents is difficult because of the size, inaccessibility, and complexity of the proteins themselves. Manipulation of the genes that control expression of these proteins, i.e., gene therapy, however, overcomes these barriers by selectively introducing recombinant DNA into tissues thereby leading to alterations in the expression of the biologically active protein in tissues. This in turn leads to alterations in cell function.
Gene therapy is now accepted as a therapeutic tool for treating diseases such as cancer. See, for example, Eck and Wilson
Goodman
&
Gilman's The Pharmacological Basis of Therapeutics
1996, Chapter 5. Gene therapy for treatment of cancer has been the focus of multiple clinical trials approved by the National Institutes of Health Recombinant DNA Advisory Committee, many of which have demonstrated successful clinical application (Hanania et al.
Am. Jour. Med.
1995 99:537-552; Johnson et al.
J. Am. Acad. Derm.
1995 32(5) :689-707; Barnes et al.
Obstetrics and Gynecology
1997 89:145-155; Davis et al.
Current Opinion in Oncology
1996 8:499-508; Roth and Cristiano
J. Natl. Canc. Inst.
1997 89(1):21-39). An important factor for success when employing alterations of gene expression as a therapeutic strategy is to begin by establishing a sound scientific basis for manipulation of that gene and its gene product.
The focus of gene therapy strategies for cancer has been not only on altering growth of a malignancy but also on diagnosis and prognosis of patients' disease. For diagnostic and prognostic methods, markers for cell cycle regulatory proteins are being developed. A goal of these methods is to decrease the morbidity and mortality of various forms of cancer by promoting earlier recognition and intervention. Early detection of tumors allows for use of more effective treatments such as immunotherapy which requires a low tumor burden (<10
9
cells). Further, more efficient classification of tumors based on molecular alterations in oncogenes, tumor suppressor genes, apoptotic genes, and/or mutator genes allows for less subjective interpretation by pathologists. This in turn leads to better choices of therapy.
Many forms of malignancy have been linked to mutations in the tumor suppressor gene known as retinoblastoma, or Rb. The retinoblastoma gene which encodes the nuclear protein p53 is the most frequently altered gene in cancer, where defects in the function of this suppressor gene lead to unregulated cell proliferation. Studies have led to the identification of an additional member of the Rb family, p107 which has also been labeled as a tumor suppressor gene. Cellular proliferation has been shown to be triggered by growth factor receptor activation as well as being controlled by cell cycle regulators such as Rb and p107 (DeCaprio et al. 1989
Cell
58:1085-1095; Zhu et al. 1993
Gene Develop.
7:1111-1125).
A third gene, Rb2/p130, has also been identified which is structurally and functionally related to both the Rb gene itself and p107 (Baldi et al.
Proc. Natl. Acad. Sci. USA
1996 93:4629-4632). All three share several regions of identity that constitute a functional domain known as the “pocket region”. This “pocket region” is involved in binding to the transforming proteins from DNA tumor viruses, such as the ElA protein from adenovirus, as well as binding to cellular transcription factors such as E2F. Therefore, the “pocket” plays a critical role in protein-protein interactions.
pRb2/p130 has been cloned and identified based upon binding to the E1A transforming domain (Mayol et al. 1993,
Oncogene
8:2561-2566). Further, the genomic structure of the human retinoblastoma-related Rb2/p130 gene which provides a molecular basis for understanding the transcriptional control of the gene itself and for delineating potential Rb2/p130 mutations in human tumors was recently disclosed (Baldi et al. 1996
Proc. Natl. Acad. Sci.
93:4629-4632). The Rb2/p130 gene has been mapped to human chromosome 16q12.2 (Yeung et al. 1993
Oncogene
8:3465-3468); deletions of this chromosome have been found in several human neoplasias including breast, hepatic, ovarian, and prostatic cancer. Accordingly, pRb2/p130 is believed to be a tumor suppressor gene in human carcinoma.
Rb2/p130 has been shown to have a role as regulator in cell cycle function. For example, Baldi et al. have shown that phosphorylation of the Rb2/p130 gene product is regulated in a cell cycle dependent manner (Baldi et al. 1995
J. Cell. Biochem.
59:402-408), in the same way that the phosphorylation of Rb is cell cycle dependent (DeCaprio et al. 1989
Cell
58:1085-1095). Further, the growth suppressive properties of the gene product of Rb2/p130 have been shown to be specific for the G
1
phase in similar fashion to pRb and p107 (Claudio et al. 1996
Cancer Res.
56:2003-2008). The gene product of Rb2/p130 has been shown to arrest growth in human tumor cell lines in a manner similar to the other members of the Rb family (i.e., pRb and p107). However, this protein also inhibits proliferation in a glioblastoma cell line that is resistant to the growth suppressant effects of both pRb and p107 (Claudio et al. 1994
Cancer Res.
54:5556-5560). Accordingly, pRb2/p130 has similar yet distinctive growth suppressive properties from pRb and p107 (Claudio et al. 1994 Cancer Res. 54:5556-5560).
It has now been found that pRb2/p130 affects tumor cell growth. The tumorigenicity mediated by a specific oncogene, c-erbB-2 (HER2\neu) is affected. Further, it has been found that pRb2/p130 affects tumor cell growth in cells not linked to expression of c-erb-2, as well as in tumor cells that are deficient in pRb2/p130 and cells that contain pRb2/p130. Compositions which comprise pRb2/p130 have been identified that can be incorporated into a vector and transfected into tumor cells to inhibit growth of the cells.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a composition comprising a vector expressing pRb2/p130 and a pharmaceutically acceptable carrier.
Another object of the present invention is to provide a method of inhibiting growth of tumor cells which comprises transfecting tumor cells with a plasmid comprising pRb2/p130.
Another object of the present invention is to provide a method for diagnostic screening of tumor cells to identify those tumors best treated by administration o
Kumar Nanda P.B.A.
McNichol, Jr. William J.
Thomas Jefferson University
Wilson Michael
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