Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1992-08-26
2002-12-10
Wang, Andrew (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S091300, C435S375000
Reexamination Certificate
active
06492512
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to methods for inhibition of growth of transformed cells, and inhibition of progression to a transformed phenotype in pre-neoplastic cells.
Transformation is a cumulative process whereby normal control of cell growth and differentiation is interrupted, usually through the accumulation of mutations affecting the expression of genes that regulate cell growth and differentiation.
Scanlon WO91/18625, WO91/18624, and WO91/18913 describes a ribozyme effective to cleave oncogene RNA from the H-ras gene. This ribozyme is said to inhibit H-ras expression in response to exogenous stimuli. Reddy WO92/00080 describes use of ribozymes as therapeutic agents for leukemias, such as CML by targeting specific portions of the BCR-ABL gene transcript.
Expression of the myc oncogene is known to alter cell growth in a number of tissues. The product of this gene is a protein which is known to be a transcriptional activator that can act singly or in combination with other oncogene proteins. The L-myc gene is often activated by translocations of DNA from other regions of the genome to the regulatory regions 5′ of the myc gene ORF. After transcription of the L-myc mRNA, alternate splicing of the transcript is known to occur. Kaye et al., 8
Mol. Cell Biol
. 196, 1988. The alternate mRNAs produced contain a common 5′ exon 1 and portions of a common exon 2. These common regions of mRNA structure allow the use of nucleic acid targeted therapeutics which can inactivate both species of mRNA with one therapeutic molecule.
SUMMARY OF THE INVENTION
The invention features use of ribozymes to inhibit the development or expression of a transformed phenotype in man and other animals by modulating expression of a gene that contributes to the expression of lung cancer. Cleavage of targeted mRNAs expressed in pre-neoplastic and transformed cells elicits inhibition of the transformed state.
Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript and efficient cleavage has been achieved in vitro. Kim et al., 84
Proc. Natl. Acad. of Sci. USA
8788, 1987, Haseloff and Gerlach, 334
Nature
585, 1988, Cech, 260
JAMA
3030, 1988, and Jefferies et al., 17
Nucleic Acid Research
1371, 1989.
Ribozymes act by first binding to a target RNA. Such binding occurs through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA which acts to cleave the target RNA. Thus, the ribozyme first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After a ribozyme has bound and cleaved its RNA target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.
This class of chemicals exhibits a high degree of specificity for cleavage of the intended target mRNA. Consequently, the ribozyme agent will only affect cells expressing that particular gene, and will not be toxic to normal tissues.
The invention can be used to treat cancer or pre-neoplastic conditions. Two preferred administration protocols can be used, either in vivo administration to reduce the tumor burden, or ex vivo treatment to eradicate transformed cells from tissues such as bone marrow prior to reimplantation.
Thus, in the first aspect the invention features an enzymatic RNA molecule (or ribozyme) which cleaves mRNA associated with development or maintenance of lung cancer, e.g., those mRNAs produced from the gene L-myc, including mRNA targets disclosed in Table 1.
TABLE 1
nucleotide number
mRNA target sequence
ID. NOS.
9
UGCAGCUCGCGCUCCCGCGCCGAUCCCGAGA
SEQ.ID.NO. 1
61
AGCGAGGGAGGGCG
SEQ.ID.NO. 2
93
GCUCGUGAGUGC
SEQ.ID.NO. 3
152
GCUGCCCCGAG
SEQ.ID.NO. 4
181
GCUCCAGGUGGC
SEQ.ID.NO. 5
200
UGGAGCGAGGGGAGCGGACAUGGACUACGACUC
SEQ.ID.NO. 6
249
ACGACUAUGACUGCGGG
SEQ.ID.NO. 7
266
GAGGAUUUCUACCGCUCCACGGC
SEQ.ID.NO. 8
289
GCCCAGCGAGGACAUCUGGA
SEQ.ID.NO. 9
327
CAUCGCCCCCCACGUC
SEQ.ID.NO. 10
391
UGGUCCCCCGGAGCCGUGGCCCG
SEQ.ID.NO. 11
414
GAGGGUGCACCGGAGA
SEQ.ID.NO. 12
445
GGGCCACUCGAAAGGCU
SEQ.ID.NO. 13
465
AGGAACUACGCCUCCAUCAUACGCC
SEQ.ID.NO. 14
490
GUGACUGCAUGUGGAGCGGCUUCUCGGCCCGGG
SEQ.ID.NO. 15
525
AACGGCUGGAGA
SEQ.ID.NO. 16
557
GCUCCUGGCGCGC
SEQ.ID.NO. 17
By “enzymatic RNA molecule” it is meant an RNA molecule which has complementarity in a substrate binding region to a specified mRNA target, and also has an enzymatic activity which is active to specifically cleave that mRNA. That is, the enzymatic RNA molecule is able to intermolecularly cleave mRNA and thereby inactivate a target mRNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA to allow the cleavage to occur. One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention.
In preferred embodiments, the enzymatic RNA molecule is formed in a hammerhead motif, but may also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RNAseP-like RNA (in association with an RNA guide sequence). Examples of such hammerhead motifs are described by Rossi et al., 8 AIDS RESEARCH AND HUMAN RETROVIRUSES 183, 1992, of hairpin motifs by Hampel et al., RNA CATALYST FOR CLEAVING SPECIFIC RNA SEQUENCES, filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, 28
Biochemistry
4929, 1989 and Hampel et al., 18
Nucleic Acids Research
299, 1990, and an example of the hepatitis delta virus motif is described by Perrotta and Been, 31
Biochemistry
16, 1992, of the RNAseP motif by Guerrier-Takada, et al., 35
Cell
849, 1983, and of the group I intron by Cech et al., U.S. Pat. No. 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic RNA molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
In a second related aspect, the invention features a mammalian cell which includes an enzymatic RNA molecule as described above. Preferably, the mammalian cell is a human cell.
In a third related aspect, the invention features an expression vector which includes nucleic acid encoding an enzymatic RNA molecule described above, located in the vector, e.g., in a manner which allo
Draper Kenneth G.
Thompson James D.
Lacourciere Karen A
McDonnell & Boehnen Hulbert & Berghoff
Ribozyme Pharmaceuticals Inc.
Wang Andrew
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