Antisense inhibition of E2A-Pbx1 expression

Details Antisense inhibition of E2A-Pbx1 expression Antisense inhibition of E2A-Pbx1 expression

2000-07-25

2003-08-19

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S377000, C536S023100, C536S024100, C536S024500, C514S04400A

Reexamination Certificate

active

06607915

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to compositions and methods for modulating expression of the E2A-Pbx1 gene which results from the fusion of the E2A and Pbx1 genes. These methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the E2A-Pbx1 gene.
BACKGROUND OF THE INVENTION
Genetic alterations are responsible for a multitude of cancers. One type of alteration is chromosomal translocation where different chromosomes are fused or where different regions within a single chromosome are fused. Both random and distinctive rearrangements can occur, with random rearrangements being common in solid tumors, while distinctive rearrangements are common in leukemias, lymphomas and sarcomas (Lengauer C., et al.,
Nature,
1998, 396, 643-649). These distinctive rearrangements involve the same breakpoints and often occur within transcription factors. The resulting transcripts can be targeted using sequence specific approaches.
Many acute leukemias are characterized by chromosomal translocations that involve transcription factors. In pediatric acute lymphoblastoid leukemia (ALL) carrying the t(1;19) chromosomal translocation, the N-terminal transactivation domain of the E2A transcription factor is fused to the homeodomain of Pbx-1 (Prl) to create the chimeric transcription factor, E2A-Pbx1 (Kamps, M. P.,
Curr. Top. Microbiol. Immunol.,
1997, 220, 25-43). This particular translocation has been found in 23% of pediatric pre-B ALL (Carroll, A., et al.,
Blood,
1984, 63, 721-724) and is associated with a poorer prognosis (Crist, W., et al.,
Blood,
1990, 76, 117-122). The resulting chimeric protein has shown transforming ability in NIH3T3 cells (Kamps, M. P., et al.,
Genes Dev.,
1991, 5, 358-368), the ability to induce malignant lymphomas in transgenic mice (Dedera, D. A., et al.,
Cell,
1993, 74, 833-843) and capable of promoting the rapid development of myeloid leukemias (Kamps, M. P. and Baltimore, D.,
Mol. Cell. Biol.,
1993, 13, 351-357).
E2A-Pbx1 physically interacts with homeodomain proteins, which are effectors of differentiation. In particular, Pbx1 interacts with Hox proteins, which are also transcription factors. Overexpression of Hox proteins can induce transformation. Pbx1 is an important contributor to differentiation, via its interaction with Hox proteins.
Expression of E2A-Pbx1 is involved in development of pre-B-cell acute lymphocytic leukemia. Expression of this protein prevents the differentiation of pre-B-cells and promotes their growth. One of the genes upregulated by E2A-Pbx-1is granulocyte colony-stimulating factor receptor (de Lau, W. B. M., et al.,
Oncogene,
1998, 17, 503-510).
Chemotherapy, including the use of methotrexate, and bone marrow transplants are the most effective treatments for leukemia and are not specific for E2A-Pbx1. There is a lack of specific inhibitors of E2A-Pbx1.
There remains a need for improved compositions and methods for inhibiting E2A-Pbx1 gene expression.
SUMMARY OF THE INVENTION
The present invention provides antisense compounds which are targeted to nucleic acids encoding E2A-Pbx1 (E2A-prl) and are capable of modulating E2A-Pbx1 mediated transformation. In one embodiment, the present invention provides oligonucleotides, including chimeric oligonucleotides, targeted to nucleic acids encoding E2A-Pbx1. The compounds of the invention are believed to be useful both diagnostically and therapeutically, and are believed to be particularly useful in the methods of the present invention.
The present invention also comprises methods of modulating E2A-Pbx1 mediated transformation, in cells and tissues, using the antisense compounds of the invention. Methods of inhibiting E2A-Pbx1 expression are provided; these methods are believed to be useful both therapeutically and diagnostically. These methods are also useful as tools, for example, for detecting and determining the role of E2A-Pbx1 in various cell functions and physiological processes and conditions and for diagnosing conditions associated with expression of E2A-Pbx1.
The present invention also comprises methods for diagnosing and treating cancers, especially pre-B-cell acute lymphocytic leukemia. These methods are believed to be useful, for example, in diagnosing E2A-Pbx1-associated disease progression. Furthermore, these methods are believed to be useful both therapeutically, including prophylactically, and as clinical research and diagnostic tools.
Thus, in a first aspect the present invention features an antisense compound having at least one modified internucleoside linkage, targeted to E2A-Pbx1 mRNA, and which inhibits the expression of the E2A-Pbx1 gene product.
In a preferred embodiment the antisense compound is an olilgonucleotide and is between 8 to 30 nucleobases in length, more preferably the antisense compound is up to 20 bases, and most preferably it is 15 to 20 bases long. In a further embodiment the modified internucleoside linkage is a phosphorothioate linkage and the nucleotide comprises a modified sugar moiety such as for example a 2′-O-methoxyethyl moiety, and/or a 5-methyl cytosine.
E2A-Pbx1 is the resulting product of the N-terminal transactivation domain of the E2A transcription factor becoming fused to the homeodomain of Pbx-1 (Prl). Thus in a preferred embodiment, the present invention features an antisense compound targeted to E2A and/or Pbx1, more preferably the antisense compound is target to bases 388-1812 of
homo sapiens
pre-B-cell leukemia transcription factor 1 (PBX1) mRNA (Genbank Accession No. NM002585) and/or bases 853-14801 of human transcription factor (E2A) mRNA (Genbank Accession number M31523). In another preferred embodiment the antisense compound is targeted to the fusion junction of E2A-Pbx1. It is also preferred that the antisense compound be contained within a pharmaceutical composition.
In an additional aspect the present invention features methods of inhibiting the expression of E2A-Pbx1 in cells or tissues. Within this method the cells or tissues are contacted with the antisense compound presently described so that expression of E2A-Pbx1 is inhibited. It is preferred that the cells or tissues are human.
In a further aspect the invention features methods for treating a human subject having a disease or condition associated with E2A-Pbx1 by administering to the subject a therapeutically or prophylactically effective amount of the antisense compound as described herein so that expression of E2A-Pbx1 is inhibited. In a preferred embodiment the disease or condition is, for example, pre-B-cell acute lymphocytic leukemia, and/or sarcomatous cancer.
Further aspects of the invention are described within the description of the preferred embodiments. The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention and from the claims.
DETAILED DESCRIPTION OF THE INVENTION
E2A-Pbx1 plays an important role in the development of leukemias, lymphomas and sarcomas. Expression of this gene is associated with a poor prognosis in pre-B-cell acute lymphocytic leukemia. As such, this chimeric protein represents an attractive target for treatment of such diseases. In particular, modulation of the expression of E2A-Pbx1 may be useful for the treatment of pre-B-cell acute lymphocytic leukemia.
The cDNA sequence of human E2A-Pbx1 (PRL) is available via Genbank Accession No. M31522. In accordance with Kamps et al. (supra) comparison of the sequence with the individual sequences for E2A and Pbx1 shows the fusion/junction point of the two genes to be at nucleotide 625 of Genbank Accession No. M31522. In a preferred embodiment the antisense compounds as described herein contain oligonucleotides targeted to the junction in E2A-Pbx1, such as for example oligonucleotides 154015, 15407, and 15409, SEQ ID NOS: 10, 11 and 12 respectively. Furthermore, due to the fusion of E2A to Pbx1, also included in the present invention are antisense compoun

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