Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1995-07-10
2000-06-27
LeGuyader, John L.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 231, 536 232, 536 2431, 536 2433, 435 6, 435 9131, 435375, C12Q 168, C07H 2104
Patent
active
060808519
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention provides ribozymes having an anchor sequence capable of anchoring the ribozyme to a region of the substrate mRNA that may be noncontiguous with the region of substrate mRNA complementary to a leg of the ribozyme. Certain preferred embodiments of the present invention include ribozymes capable of cleaving the L6fusion mRNA or both the L6 and K28 mRNAs expressed by the hematopoietic cells of some CML and ALL patients. The present invention also furnishes methods of treating leukemias using oligonucleotide therapeutics.
BACKGROUND OF THE INVENTION
The potential use of ribozymes as pharmaceuticals is an exciting prospect. An important requirement for the development of ribozyme pharmaceutical products is the ability to specifically target a ribozyme to a cellular RNA of interest. This can be especially difficult when designing a ribozyme for a chimeric RNA molecule that is homologous to a second RNA molecule, particularly if cleavage of the second RNA is detrimental to the host. It may be equally difficult to target an RNA molecule that is folded in a way that prevents ribozyme interactions at or near the ribozyme cleavage site. We have encountered both of these problems in our attempts to design ribozymes that are specific for an aberrant mRNA associated with chronic myelogenous leukemia (CML).
Chronic Myelogenous Leukemia (CML) and acute lymphocytic leukemia (ALL) represent two different types of leukemias. CML is a chronic myeloproliferation disorder associated with the cytogenic marker called the Philadelphia chromosome (Nowell, P. C. and Hungerford, D. A., Science 1960, 132, 1497) in approximately 95% of patients. The Philadelphia chromosome is a chromosomal abnormality resulting from reciprocal translocations between chromosomes 9 and 22 (Mercola, M. et al., Science 1933, 221, 663).
The breakpoints on chromosome 22 are clustered in a 6 Kb region termed the breakpoint cluster region (bcr) (Groffen, J. et al., Cell 1984, 36, 93-99), while on chromosome 9, the breakpoints are scattered throughout a 90 Kb region upstream from c-abl exon 2 (Heisterkamp, N. et al., Nature 1983, 306, 239-242). The resultant fusion transcripts, which are about 8.5 kb long, contain bcr sequences upstream and abl sequences downstream.
The cellular gene abl, a highly conserved gene, represents the progenitor of the viral transforming gene (v-abl) of Abelson leukemia virus. v-abl confers to Abelson leukemia virus the ability to transform a broad range of hematopoietic cell types. Transformation is mediated by a tyrosine kinase encoded by the viral genome, composed of v-abl polypeptide attached at its N-terminus to viral gag polypeptide. The human abl gene was mapped to chromosome 9, and is expressed as a 145 kd protein having tyrosine kinase activity. Misregulation of abl is implicated in CML in humans. Shtivelman et al., Cell 1986, 47, 277-284.
The various 9:22 translocations associated with the Philadelphia chromosome can be subdivided into two types: K28 translocations and L6 translocations. In the K28 mRNA, abl exon 2 is linked to bcr exon 3. In the L-6 mRNA, abl exon 2 is linked to bcr exon 2. C-myc mRNA is pertinent for blast crisis in CML.
A third type of 9:22 translocation has also been identified. The chromosome 9 breakpoints specific for this type of translocation are located 5' of the L6 breakpoints. The presence of this abnormal chromosome, however, is associated with the establishment of acute lymphocytic leukemia (ALL) not CML. Selleri et al., Blood 1990, 75, 1146-1153. Further, the K28 and L6 mRNAs have been associated with some patients with ALL.
Much emphasis has been placed on the role of mRNA K28 and the establishment of CML. (Shtivelman, E. et al., Cell 1986, 47, 277-284; Kubonishi, I. and Miyoshi, I., Int. J. Cell Cloning 1983, 1, 105-117; Shtalrid, M. T. et al., Blood 1988, 72, 485-490; Mills, K. I. et al., Blood 1988, 72, 1237-1241).
In the K28 translocations, the chromosomal 22 breakpoints lie between bcr exons 3 and 4 (Shtivelman, E. et al., Cell 1986, 47, 277
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Coney Leslie R.
Oakes Fred T.
Pachuk Catherine J.
American Home Products Corporation
LeGuyader John L.
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