Antisense IAP nucleic acids and uses thereof

Details Antisense IAP nucleic acids and uses thereof Antisense IAP nucleic acids and uses thereof

2000-09-28

2004-01-06

McGarry, Sean (Department: 1635)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S024300, C536S024310, C536S024330, C435S091310

Reexamination Certificate

active

06673917

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to antisense IAP nucleic acids and methods of using them to increase apoptosis.
BACKGROUND OF THE INVENTION
One way by which cells die is referred to as apoptosis, or programmed cell death. Apoptosis often occurs as a normal part of the development and maintenance of healthy tissues. The process may occur so rapidly that it is difficult to detect.
The apoptosis pathway is now known to play a critical role in embryonic development, viral pathogenesis, cancer, autoimmune disorders, and neurodegenerative diseases, as well as other events. The failure of an apoptotic response has been implicated in the development of cancer, autoimmune disorders, such as lupus erythematosis and multiple sclerosis, and in viral infections, including those associated with herpes virus, poxvirus, and adenovirus.
Baculoviruses encode proteins that are termed inhibitors of apoptosis (IAPs) because they inhibit the apoptosis that would otherwise occur when insect cells are infected by the virus. These proteins are thought to work in a manner that is independent of other viral proteins. The baculovirus IAP genes include sequences encoding a ring zinc finger-like motif (RZF), which is presumed to be directly involved in DNA binding, and two N-terminal domains that consist of a 70 amino acid repeat motif termed a BIR domain (Baculovirus IAP Repeat).
The role of apoptosis in cancer has only recently been appreciated. The identification of growth promoting “oncogenes” in the late 1970's gave rise to an almost universal focus on cellular proliferation that dominated research in cancer biology for many years. Long-standing dogma held that anti-cancer therapies preferentially targeted rapidly dividing cancer cells relative to “normal” cells. This explanation was not entirely satisfactory, since some slow growing tumors are easily treated, while many rapidly dividing tumor types are extremely resistant to anti-cancer therapies. Progress in the cancer field has now led to a new paradigm in cancer biology wherein neoplasia is viewed as a failure to execute normal pathways of programmed cell death. Normal cells receive continuous feedback from their neighbors through various growth factors, and commit “suicide” if removed from this context. Cancer cells somehow ignore these commands and continue inappropriate proliferation. Cancer therapies, including radiation and many chemotherapies, have traditionally been viewed as causing overwhelming cellular injury. New evidence suggests that cancer therapies actually work by triggering apoptosis.
Both normal cell types and cancer cell types display a wide range of susceptibility to apoptotic triggers, although the determinants of this resistance are only now under investigation. Many normal cell types undergo temporary growth arrest in response to a sub-lethal dose of radiation or cytotoxic chemical, while cancer cells in the vicinity undergo apoptosis. This provides the crucial treatment “window” of appropriate toxicity that allows successful anti-cancer therapy. It is therefore not surprising that resistance of tumor cells to apoptosis is emerging as a major category of cancer treatment failure.
Compared to the numerous growth-promoting oncogenes identified to date (>100), relatively few genes have been isolated that regulate apoptosis. The Bcl-2 gene was first identified as an oncogene associated with the development of follicular lymphomas. In contrast to all other oncogenes identified to date, Bcl-2 displays no ability to promote cell proliferation, and instead has been demonstrated to suppress apoptosis by a variety of triggers. Elevated Bcl-2 expression is associated with a poor prognosis in neuroblastoma, prostate and colon cancer, and can result in a multidrug resistant phenotype in vitro. Although the study of Bcl-2 has helped revolutionize cancer paradigms, the vast majority of human malignancies do not demonstrate aberrant Bcl-2 expression.
In contrast to the findings with Bcl-2, mutation of the p53 tumor suppresser gene has been estimated to occur in up to 50% of human cancers and is the most frequent genetic change associated with cancer to date. The p53 protein plays a crucial role in surveying the genome for DNA damage. The cell type and degree of damage determines whether the cell will undergo growth arrest and repair, or initiate apoptosis. Mutations in p53 interfere with this activity, rendering the cell resistant to apoptosis by a wide range of cellular insults. Some progress has been made in understanding the molecular biology of p53, but many questions remain. p53 is known to function as a transcription factor, with the ability to positively or negatively regulate the expression of a variety of genes involved in cell cycle control, DNA repair, and apoptosis (including the anti-apoptotic Bcl-2 gene described above and the related pro-apoptotic gene Bax). The drug resistant phenotype conferred by p53 alterations has been linked to Bcl-2/Bax regulation, but this correlation does not hold for most cancer types, leaving open the possibility that other critical genes regulated by p53 remain to be identified.
SUMMARY OF THE INVENTION
We have discovered that inhibitor of apoptosis (IAP) protein overexpression is associated with a wide range of cancer types including ovarian cancer, adenocarcinoma, lymphoma, and pancreatic cancer. In addition, we have found that nuclear localization, fragmentation of the IAPs, and overexpression of the IAPs in the presence of p53 mutations correlate with a cancer diagnosis, a poor prognosis, and resistance to numerous chemotherapeutic cancer drugs. These discoveries provide diagnostic, prognostic, and therapeutic compounds and methods for the detection and treatment of proliferative diseases. One way in which the expression of an IAP in a cell can be decreased is by administering to the cell a negative regulator of the IAP apoptotic pathway, for example, an antisense nucleic acid.
In general, the invention features methods and reagents useful for inducing apoptosis in a cell. The methods and reagents of the invention are useful in treating cancers, and other proliferative diseases.
In a first aspect, the invention features an inhibitor of apoptosis (IAP) antisense nucleic acid that inhibits IAP biological activity, regardless of the length of the antisense nucleic acid. In preferred embodiments, the IAP is XIAP, HIAP1, or HIAP2. In other preferred embodiments, the antisense nucleic acid is mammalian, for example, mouse or human. In yet another embodiment, the antisense nucleic acid is between 8 and 30 nucleotides in length.
In still other further preferred embodiments, the XIAP antisense is chosen from any one of SEQ ID NOS: 1 through 96, and the HIAP1 antisense is chosen from any one of SEQ ID NOS: 97 through 194. Preferably the IAP biological activity is inhibition of apoptosis or inhibition of IAP RNA or polypeptide expression. The antisense nucleic acid may comprise at least one modified internucleoside linkage. Preferably the modified internucleoside linkage is a phosphorothioate, a methylphosphonate, a phosphotriester, a phosphorodithioate, or a phosphoselenate linkage. In addition, the antisense nucleic acid may comprise at least one modified sugar moiety. Preferably this modified sugar moiety is a 2′-O methoxyethyl group or a 2′-O methyl group. In still another preferred embodiment, the antisense nucleic acid is a chimeric nucleic acid. Preferably the chimeric nucleic acid comprises DNA residues linked together by phosphorothioate linkages, and the DNA residues are flanked on each side by at least one 2′-O methyl RNA residue linked together by a phosphorothioate linkage. More preferably the DNA residues are flanked on each side by at least three 2′-O methyl RNA residues. In yet another embodiment, the antisense nucleic acid is a ribozyme.
In a second aspect, the invention features a method of enhancing apoptosis in a cell, involving administering to the cell a negative regulator of the IAP-dependent antiapoptotic pathway. In preferred embodime

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