Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1994-02-07
2003-04-08
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S091100, C435S091300, C435S091310, C435S320100, C435S325000, C536S023100, C536S023100
Reexamination Certificate
active
06544755
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.
c-Myc, when activated, can induce malignancy in a variety of tissues, most notably hematopoietic tissues (Leder et al., 222
Science
765, 1983). The most common mechanism of c-myc activation is translocation to any of the immunoglobulin (Ig) or T cell receptor loci during lymphoid maturation (Croce and Nowell, 65
Blood
1, 1985; Klein and Klein, 6
Immunol. Today
208, 1985). For example, in Burkitt's lymphoma the c-myc locus on chromosome 8 translocates most often to the Ig heavy chain locus on chromosome 14, but also to the lambda or kappa light chain Ig genes on chromosomes 2 and 22 (Magrath, in “Epstein-Barr Virus and Associated Diseases”, M. Nijhoff Publishing:631, 1986). In some instances the c-myc transcription region is altered in the non-coding exon 1 region; in such cases transcription is initiated at a cryptic promoter present in the first intron of the c-myc locus. These rearrangements are thought to lead to deregulation of c-myc expression.
c-Myc is not normally expressed in quiescent cells, but is temporally expressed in actively-dividing cells, most prominently during transition from G0 to G1 phases of growth induction.
Experiments with transfected cell lines and transgenic animals have shown that c-myc activation plays a critical role, but is not sufficient for transformation (Adams et al., 318
Nature
533, 1985; Lombardi et al., 49
Cell
161, 1987; Schwartz et al., 6
Mol. Cell. Biol.
3221, 1986; Langdon et al., 47
Cell
11, 1986). Targeted inhibition of c-myc expression in tumor cell lines using antisense oligonucleotides has shown that c-myc expression is required for growth in certain lymphomas (McManaway et al., 335
Lancet
808, 1990).
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 Burkitt's lymphoma, acute lymphocytic leukemia and other neoplastic conditions. 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. Nat. 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 Burkitt's lymphoma or acute lymphocytic leukemia, e.g., those mRNAs produced from the gene c-myc, including mRNA targets disclosed in Table 1.
TABLE 1
nucleotide
mRNA target sequence
ID. NO.
(469)
CAGGACCCGCUUCUCUGAAAGGCUCUCCUU
SEQ.ID.NO.1
(553)
GCGACGAUGCCCCUCAACGUUAGCU
SEQ.ID.NO.2
(589)
AACUAUGACCUCGACUACGACUCGGU
SEQ.ID.NO.3
(629)
ACUGCGACGAGGAGGAGAACUUCUACCA
SEQ.ID.NO.4
(662)
AGCAGCAGAGCGA
SEQ.ID.NO.5
(680)
AGCCCCCGGCGCCCAGCGAGGAUAUCUGGA
SEQ.ID.NO.6
(725)
UGCCCACCCCGCC
SEQ.ID.NO.7
(758)
CCGGGCUCUGCUCGCCCUCCUA
SEQ.ID.NO.8
(783)
UGCGGUCACACCCU
SEQ.ID.NO.9
(813)
CAACGACGGCGGUGGCGGGAGCUUCUCCACGGCCGACCA
SEQ.ID.NO.10
(876)
GGGAGGAGACAU
SEQ.ID.NO.11
(907)
UGCGACCCGGACGACGAGACCUUCAUCAAAAACAUCAUCA
SEQ.ID.NO.12
(981)
CGCCAAGCUCGUCUCAGAGAAGCUGGCCUCCUACCA
SEQ.ID.NO.13
(1023)
GCGCAAAGACAGCG
SEQ.ID.NO.14
(1043)
CGAACCCCGCCC
SEQ.ID.NO.15
(1077)
CUCCAGCUUGUACCUGCAGGA
SEQ.ID.NO.16
(1114)
UCAGAGUGCAUCGACCC
SEQ.ID.NO.17
(1124)
UCGACCCCUCGGUGGUCUUCCCCUACCCUCUCAACGAC
SEQ.ID.NO.18
(1168)
UCGCCCAAGUCCUGCGCCUCGCAAGACUCCAGCGC
SEQ.ID.NO.19
(1230)
CUCCUCGACGGA
SEQ.ID.NO.20
(1258)
AGCCCCGAGCCCC
SEQ.ID.NO.21
(1276)
CUCCAUGAGGAGA
SEQ.ID.NO.22
(1357)
GUGGAAAAGAGG
SEQ.ID.NO.23
(1376)
CUGGCAAAAGGUCA
SEQ.ID.NO.24
(1397)
GAUCACCUUCUGCUGGAGGCCACAGCAAACCUCCUCACA
SEQ.ID.NO.25
(1459)
CACGUCUCCACACAUCAGCACAACUACGCA
SEQ.ID.NO.26
(1496)
CCUCCACUCGGAAGGACUAUCC
SEQ.ID.NO.27
(1523)
CCAAGAGGGUCAAGUUGGA
SEQ.ID.NO.28
(1547)
UCAGAGUCCUGAGACA
SEQ.ID.NO.29
(1569)
CAACAACCGAAAAUGCA
SEQ.ID.NO.30
(1643)
UGGAGCGCCAGAGG
SEQ.ID.NO.31
(1662)
CGAGCUAAAACGGAGCU
SEQ.ID.NO.32
(1684)
GCCCUGCGUGACCAGAUCCCGGA
SEQ.ID.NO.33
(1712)
AAAACAAUGAAAAGGCCCCCAAGGUAGUUAUCCUUAAAA
SEQ.ID.NO.34
(1755)
CACAGCAUACAUCCUGUCCGUCCAAG
SEQ.ID.NO.35
(1786)
GAGCAAAAGCUCAU
SEQ.ID.NO.36
(1805)
AAGAGGACUUGUUGCGGAAACGACGAGAACAGUUGAAAC
SEQ.ID.NO.37
(1845)
CAAACUUGAACAGCUACGGAACUCUUGUGCGU
SEQ.ID.NO.38
(1883)
AAGUAAGGAAAACGA
SEQ.ID.NO.39
(1904)
CUAACAGAAAUGUCCUGAGCAAUCACCUAUGAACU
SEQ.ID.NO.40
(1964)
ACCUCACAACCU
SEQ.ID.NO.41
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 spec
Draper Kenneth G.
Thompson James D.
McDonnell & Boehnen Hulbert & Berghoff
McGarry Sean
Ribozyme Pharmaceuticals Inc.
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