Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1997-08-13
2001-06-26
Clark, Deborah J. (Department: 1633)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S320100, C536S023500
Reexamination Certificate
active
06251871
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of tumor biology. In particular, the invention relates to a nucleic acid encoding a tumor suppressor and its use in inhibiting tumor growth. In one embodiment, the invention relates to expression constructs encoding p16 and their use in inhibiting cancer.
2. Description of the Related Art
Cancer is one of the leading causes of human disease, being responsible for 526,000 deaths in the United States each year (Boring et al., 1993). Lung cancer alone kills more than 140,000 people annually in the United States. Recently, age-adjusted mortality from lung cancer has surpassed that from breast cancer in women. Although implementation of smoking-reduction programs has decreased the prevalence of smoking, lung cancer mortality rates will remain high well into the twenty-first century. Unfortunately, current treatment methods for cancer, including radiation therapy, surgery and chemotherapy, are known to have limited effectiveness. The rational development of new therapies for lung cancer largely will depend on gaining an improved understanding of the biology of cancer at the molecular level.
With advances in molecular genetics and biology, it has become evident that altered expression of normal genes can lead to the initiation of transforming events that result in the creation of cancer cells. The conventional therapy for malignancy, such as chemotherapy and radiation, has focused on mass cell killing without specific targeting, often resulting in damaging side effects. A new direction in cancer therapy is to deliver a normal gene to replace or correct the mutated gene, thereby altering the malignant phenotype of transformed cells. Several expression constructs have been developed in order to deliver a gene into somatic cells with high efficiency.
Cells are regulated in both positive (stimulatory) and negative (suppressive) manners. Loss of negative regulation of cell growth is often found in malignant cells. Accumulating molecular genetic evidence has revealed that loss of negative regulators, or increase in positive regulators in normal cells, can produce such cellular growth abnormalities. Most negative regulators (Marx, 1993; Grunicke and Maly, 1993), referred to as tumor suppressors, have been found to be involved either in direct control of the cell cycle (e.g., Rb, p53, WT-1) or in the signaling pathway leading to cell growth and differentiation (e.g., NF-1). In addition, recent data suggest that genes related to the maintenance of cell architecture and polarity also may function as tumor suppressors (Marx, 1993; Fearon et al., 1990; Trofatter et al., 1993).
The major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4 (CDK4), regulates progression through the G
1
. The activity of this enzyme may be to phosphorylate Rb at late G
1
. The activity of CDK4 is controlled by an activating subunit, D-type cyclin, and by an inhibitory subunit, the p16
INK4
protein. p16
INK4
has been biochemically characterized as a protein that specifically binds to and inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et al., 1993; Serrano et al., 1995). Since the p16
INK4
protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4, resulting in hyperphosphorylation of the Rb protein. p16 also is known to regulate the function of CDK6.
p16
INK4
belongs to a newly described class of CDK-inhibitory proteins that also includes p15
B
, p21
WAF1
, and p27
KIP1
. The p16
INK4
gene maps to 9p21, a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the p16
INK4
gene are frequent in human tumor cell lines. This evidence suggests that the p16
INK4
gene is a tumor suppressor gene. This interpretation has been challenged, however, by the observation that the frequency of the p16
INK4
gene alterations is much lower in primary uncultured tumors than in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994; Hussussian et al., 1994; Kamb et al., 1994; Kamb et al., 1994; Mori et al., 1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et al., 1994; Arap et al., 1995). Restoration of wild-type p16
INK4
function by transfection with a plasmid expression vector reduced colony formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).
SUMMARY OF THE INVENTION
The present invention addresses the need for improved therapy for lung and other p16-associated cancers by providing expression constructs containing a nucleic acid encoding p16. It also is an object of the present invention to provide methods for the use of such compositions and, in particular, use in the treatment of cancer. In another embodiment, the present invention encompasses methods for transforming cells using a p16 nucleic acid in an expression construct.
The present invention also encompasses expression constructs that comprise a promoter functional in eukaryotic cells and a nucleic acid encoding p16, the nucleic acid being under transcriptional control of the promoter.
In a preferred embodiment, the expression constructs further comprise a polyadenylation signal. In another embodiment, the constructs further comprise a selectable marker. In a further embodiment, the expression construct is an adenovirus. In a preferred embodiment, the expression construct is an adenovirus that lacks at least a portion of the E1 region.
In certain embodiments, the nucleic acid is a cDNA. In other embodiments the nucleic acid is a genomic DNA. Still other embodiments include a combination of cDNA and genomic DNA, for example, in a mini-gene construct. In an exemplary embodiment the nucleic acid is positioned in a sense orientation with respect to said promoter. In another embodiment the nucleic acid is positioned in an antisense orientation.
The present invention also includes pharmaceutical compositions comprising an expression construct with a promoter functional in eukaryotic cells and a nucleic acid encoding p16, along with a pharmaceutically acceptable buffer, solvent or diluent. In certain embodiments, the expression construct and pharmaceutically acceptable buffer, solvent or diluent are supplied in a kit.
The invention also provides a method for restoring proper p16 function in a cell that lacks p16 function or expresses a functional p16 that is improperly compartmentalized. This method comprises contacting such a cell with an expression construct as described above, wherein the nucleic acid is positioned in a sense orientation. In an exemplary embodiment of the invention, the cell is a transformed cell and the contacting reverses the transformed phenotype. In a further embodiment, the cell is a lung, bladder, leukemia or melanoma cancer cell and, in still a further embodiment, the expression construct is an adenovirus.
The present invention further comprises a method for inhibiting p16 function in a cell. This method comprises contacting such a cell with an expression construct as described above, wherein the nucleic acid is positioned in an antisense orientation. In a further embodiment, the expression construct is an adenovirus.
Another embodiment of the invention is a method of treating a mammal with cancer. This method comprises administering to an animal a pharmaceutical composition comprising an expression construct having a promoter functional in eukaryotic cells and a nucleic acid encoding p16, positioned in a sense orientation, in a pharmaceutically acceptable buffer, solvent or diluent. In a particular embodiment of the invention, the mammal is a human. In another embodiment, administering is via intravenous injection. In a further embodiment, the cancer is lung cancer.
In further embodiments the present invention encompasses methods for detecting cancer cells in a sample by detecting p16 or a nucleic acid encoding a p16.
REFERENCES:
patent: 5399346 (1995-03-01), Anderson et al.
patent: 5624819 (1997-04-01), Skolnick et al.
patent: 56
Jin Xiaomei
Roth Jack A.
Board of Regents , The University of Texas System
Clark Deborah J.
Fulbright & Jaworski
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