AU710972B2 - Oligonucleotide modulation of protein kinase C - Google Patents

Oligonucleotide modulation of protein kinase C Download PDF

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AU710972B2
AU710972B2 AU70071/98A AU7007198A AU710972B2 AU 710972 B2 AU710972 B2 AU 710972B2 AU 70071/98 A AU70071/98 A AU 70071/98A AU 7007198 A AU7007198 A AU 7007198A AU 710972 B2 AU710972 B2 AU 710972B2
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oligonucleotide
seq
protein kinase
mrna
gene
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C. Frank Bennett
Russell T Boggs
Nicholas M Dean
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Ionis Pharmaceuticals Inc
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Isis Pharmaceuticals Inc
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Description

OLIGONUCLEOTIDE MODULATION OF PROTEIN KINASE
C
FIELD OF THE INVENTION This invention relates to therapies, diagnostics, and research reagents for disease states which respond to 5 modulation of the expression of protein kinase C. In particular, this invention relates to antisense oligonucleotides specifically hybridizable with nucleic acids relating to protein kinase C. These oligonucleotides have been found to modulate the expression of protein kinase C. Palliation and therapeutic effect result.
BACKGROUND OF THE INVENTION The phosphorylation of proteins plays a key role in the transduction of extracellular signals into the cell.
The enzymes, called kinases, which effect such phosphorylations are targets for the action of growth factors, hormones, and other agents involved in cellular metabolism, proliferation and differentiation. One of the major signal transduction pathways involves the enzyme protein kinase C (PKC), which is known to have a critical influence on cell proliferation and differentiation.
PKC
is activated by diacylglycerols (DAGs), which are metabolites released in signal transduction.
:Interest in PKC was stimulated by the finding that PKC is the major, and perhaps only, cellular receptor through which a class of tumor-promoting agents called phorbol esters exert their pleiotropic effects on cells [Gescher et al., Anti-Cancer Drug Design 4:93-105 (1989)].
Phorbols capable of tumor production can mimic the effect 2 of DAG in activating PKC, suggesting that these tumor promoters act through PKC and that activation of this enzyme is at least partially responsible for the resulting tumorigenesis [Parker et al., Science 233:853-866 (1986)].
Experimental evidence indicates that PKC plays a role in growth control in colon cancer. It is believed that specific bacteria in the intestinal tract convert lipids to DAG, thus activating PKC and altering cell proliferation. This may explain the correlation between high dietary fat and colon cancer [Weinstein, Cancer Res.
(Suppl.) 5 1:5080s-5085s (1991)]. It has also been demonstrated that a greater proportion of the PKC in the colonic mucosa of patients with colorectal cancer is in an activated state compared to that of patients without cancer 15 [Sakanoue et al., Int. J. Cancer 48:803-806 (1991)].
Increased tumorigenicity is also correlated with overexpression of PKC in cultured cells inoculated into nude mice. A mutant form of PKC induces highly malignant tumor cells with increased metastatic potential.
20 Sphingosine and related inhibitors of PKC activity have been shown to inhibit tumor cell growth and radiationinduced transformation in vivo [Endo et al., Cancer Research 51:1613-1618 (1991); Borek et al., Proc. Natl.
Acad. Sci. 88:1953-1957 (1991)]. A number of experimental or clinically useful anti-cancer drugs show modulatory effects on PKC. Therefore, inhibitors of PKC may be important cancer-preventive or therapeutic agents. PKC has been suggested as a plausible target for more rational design of conventional anti-cancer drugs [Gescher, A. and Dale, Anti-Cancer Drug Design, 4:93-105 (1989)].
Experiments also indicate that PKC plays an important role in the pathophysiology of hyperproliferative skin disorders such as psoriasis and skin cancer.
Psoriasis is characterized by inflammation, hyperproliferation of the epidermis and decreased differentiation of cells. Various studies indicate a role for PKC in causing these symptoms. PKC stimulation in 3 cultured keratinocytes can be shown to cause hyperproliferation. Inflammation can be induced by phorbol esters and is regulated by PKC. DAG is implicated in the involvement of PKC in dermatological diseases, and is formed to an increased extent in psoriatic lesions.
Inhibitors of PKC have been shown to have both antiproliferative and antiinflammatory effects in vitro.
Some antipsoriasis drugs, such as cyclosporine A and anthralin, have been shown to inhibit PKC. Inhibition of PKC has been suggested as a therapeutic approach to the treatment of psoriasis [Hegemann, L. and G. Mahrle, Pharmacology of the Skin, H. Mukhtar, ed., p. 357-368,
CRC
Press, Boca Raton, FL, 1992].
PKC is not a single enzyme, but a family of 15 enzymes. At the present time at least seven isoforms (isozymes) of PKC have been identified: a, f, 7, 6, E, and r. These isozymes have distinct patterns of tissue and S**o organ localization (see Nishizuka, Nature, 334:661-665 (1988) for review) and may serve different physiological 20 functions. For example, PKC-y seems to be expressed only S" in the central nervous system. PKC-a and -9 are expressed in most tissues, but have different patterns of expression in different cell types. For example, both PKC-a and PKC-I are expressed in, and have been purified from, human 25 epidermis. While PKC-a has been detected mainly in keratinocytes of the basal layers of the epidermis, PKC-f is found mainly in the middle layers of the epidermis and Langerhans cells. PKC-T has been found predominantly in the skin and lungs, with levels of expression much higher in these tissues than in the brain. This is in contrast to other members of the PKC family which tend to be most abundantly expressed in the brain [Osada et al., J. Biol.
Chem. 265:22434-22440 (1990)]. Another PKC isozyme, PKC-r, is believed to play a critical role in control of proliferative cascades. This was demonstrated by using antisense RNA, peptide inhibitors or a phosphorothioate antisense oligonucleotide targeted to the 4 AUG of Xenopus PKC- to deplete PKC-r levels in Xenopus oocytes. These depleted oocytes were shown to be resistant to maturation in response to insulin, while the maturation pathway activated by progesterone was not affected.
WO
93/20101. While the PKC isozymes listed here are preferred for targeting by the present invention, other isozymes of PKC are also comprehended.by the present invention.
It is presently believed that different PKC isozymes may be involved in various disease processes depending on the organ or tissue in which they are expressed. For example, in psoriatic lesions there is an alteration in the ratio between PKC-a and PKC-S, with preferential loss of PKC-1 compared to normal skin [Hegemann, L. and G. Mahrle, Pharmacology of the Skin, H. Mukhtar, ed., p. 357-368,
CRC
15 Press, Boca Raton, FL, 1992].
Even for a given isozyme, there may be multiple
RNA
.transcripts expressed from a single gene. In the case of PKCa, for example, two mRNA transcripts are seen: a long (approximately 8.5 kb) transcript and a short (approximately 4 kb) transcript. Multiple PKCa transcripts are produced from the murine and the bovine PKCa genes as well. The ratio between the long and short transcriDts varies between species and is believed to vary between tissues as well. In addition, there may be some 25 correlation between this ratio and the proliferative state Sof cells.
Although numerous compounds have been identified as PKC inhibitors (see Hidaka and Hagiwara, Trends in Pharm.
Sci. 8:162-164 (1987) for review), few have been found which inhibit PKC specifically. While the quinoline sulfonamide derivatives such as 2 -methylpiperazine inhibit PKC at micromolar concentrations, they exhibit similar enzyme inhibition kinetics for PKC and the CAMP-dependent and cGMP-dependent protein kinases. Staurosporine, an alkaloid product of Streptomyces sp., and its analogs, are the most potent in vitro inhibitors of PKC identified to date. However, they exhibit only limited selectivity among different protein kinases [Gescher, Anti-Cancer Drug Design 4:93-105 (1989)]. Certain ceramides and sphingosine derivatives have been shown to have PKC inhibitory activity and to have promise for therapeutic uses, however, there remains a long-felt need for specific inhibitors of the enzymes.
There is also a desire to inhibit specific PKC isozymes, both as a research tool and as treatment for diseases which may be associated with particular isozymes. Godson et a. Biol. Chem. 268:11846-11950 (1993)] recently disclosed use of stable transfection of antisense PKC-a cDNA in cytomegalovirus promotor-based expression vectors to specifically decrease expression of PKC-ca protein by approximately 70%. It was demonstrated that this inhibition causes a loss of phospholipase
A
2 -mediated arachidonic acid release in response to the phorbol ester PMA. Attempts by the same researchers at inhibiting PKC activity with oligodeoxynucleotides were 15 ultimately unsuccessful due to degradation of oligonucleotides.
SUMMARY OF THE INVENTION The present invention provides an oligonucleotide having up to nucleotide units specifically hybridizable with a protein kinase C-e gene or 20 mRNA.
The present invention further provides an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-a gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:52 and 53.
25 The present invention further provides an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-r gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:54, 55, 56, 57, 58.
59. 60, 61, and 62.
The present invention further provides an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-c gene or mRNA. wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:63. 64. 67. 68. 69.
71. 73. 74. 76, 77. 78. 79. 80. 81. 84. and In a preferred embodiment at least one of the intersugar linkages between nucleotide units of the oligonucleotides is a phosphorothioate.
-6- Preferably, at least one of the nucleotide units comprises a modification at the 2' position of the sugar. The modification may be a 2'-Oalkyl or 2'-fluoro modification. More preferably, the modification is a methyl or 2 '-O-propxyl modification.
The present invention further provides pharmaceutical compositions comprising an oligonucleotide of the present invention and a pharmaceutically acceptable carrier or diluent.
The present invention also provides a method of modulating the expression of protein kinase C in cells comprising contacting the cells with an oligonucleotide having up to about 50 nucleotide units, said oligonucleotide being specifically hybridizable with a protein kinase C-e gene or mRNA.
The present invention also provides a method of modulating the expression of protein kinase C in cells comprising contacting the cells with 15 an oligonucleotide having up to about 50 nucleotide units, said oligonucleotide being specifically hybridizable with a protein kinase C-a gene or mRNA. wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:52 and 53.
The present invention also provides a method of modulating the 20 expression of protein kinase C in cells comprising contacting the cells with an oligonucleotide having up to about 50 nucleotide units. said oligonucleotide being specifically hybridizable with a protein kinase C-q gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:54, 55, 56, 57. 58.
25 59, 60, 61, and 62.
i The present invention also provides a method of modulating the expression of protein kinase C in cells comprising contacting the cells with an oligonucleotide having up to about 50 nucleotide units. said oligonucleotide being specifically hybridizable with a protein kinase C- gene or mRNA. wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:63. 64. 67. 68. 69, 71. 73.
74. 76. 77, 78. 79. 80. 81. 84. and oligonucleotide comprises a modification on the 2' position of the sugar.
The present invention also provides a method of detecting in a sample the presence of a protein kinase C-e gene or rnRNA comprising contacting the sample with an oligonucleotide having up to 50 nucleotide 6Aunits specifically hybridizable with said gene or mRNA, and detecting hybridization.
The present invention also provides a method of detecting in a sample the presence of a protein kinase C-a gene or mRNA comprising contacting the sample with an oligonucleotide having up to 50 nucleotide units specifically hybridizable with said gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:52 and 53, and detecting hybridization.
The present invention also provides a method of detecting in a sample the presence of a protein kinase C-q gene or mRNA comprising contacting the sample with an oligonucleotide having up to 50 nucleotide units specifically hybridizable with said gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:54, 55, 56, 57, 58, 59, 60, 61, and 62, and detecting hybridization.
The present invention also provides a method of detecting in a sample the presence of a protein kinase C- gene or mRNA comprising contacting the sample with an oligonucleotide having up to 50 nucleotide units specifically hybridizable with said gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 63, 64, 67, 68. 69, 71, 73, 74, 76. 77, 78. 79. 80. 81.
84. and 85. and detecting hybridization.
The present invention also provides a method of treating a condition associated with expression of protein kinase C comprising administering to a 25 mammal or cells thereof with a therapeutically effective amount of an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-e gene or mRNA.
The present invention also provides a method of treating a condition associated with expression of protein kinase C comprising administering to a mammal or cells thereof with a therapeutically effective amount of an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-ce gene or mRNA. wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:52 and 53.
The present invention also provides a method of treating a condition associated with expression of protein kinase C comprising administering to a 6Bmammal or cells thereof with a therapeutically effective amount of an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-77 gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:54, 55, 56, 57, 58, 59, 60, 61, and 62.
The present invention also provides a method of treating a condition associated with expression of protein kinase C comprising administering to a mammal or cells thereof with a therapeutically effective amount of an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C- gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:63, 64, 67, 68, 69, 71, 73, 74, 76, 77, 78, 79, 80, 81, 84, and The present invention also provides a method of diagnosing a condition associated with protein kinase C comprising contacting a sample 15 from a mammal suspected of having a condition associated with protein kinase C with an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-E gene or mRNA, and detecting hybridization.
The present invention also provides a method of diagnosing a condition associated with protein kinase C comprising contacting a sample from a mammal suspected of having a condition associated with protein kinase C with an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-c gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the 25 group consisting of SEQ ID NO: 52 and 53, and detecting hybridization.
The present invention also provides a method of diagnosing a condition associated with protein kinase C comprising contacting a sample from a mammal suspected of having a condition associated with protein kinase C with an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-q gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group;consisting of SEQ ID NO:54. 55, 56, 57. 58. 59, 60, 61. and 62. and detecting hybridization.
The present invention also provides a method of diagnosing a condition associated with protein kinase C comprising contacting a sample from a mammal suspected of having a condition associated with protein 6Ckinase C with an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C- gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:63, 64, 67, 68, 69, 71, 73, 74, 76, 77, 78, 79, 80, 81, 84, and 85, and detecting hybridization.
The present invention also provides an isolated nucleic acid molecule comprising a sequence substantially homologous to the sequence set forth in SEQ ID NO:105.
The present invention also provides an antisense oligonucleotide up to 50 nucleotides in length comprising a nucleotide sequence specifically hybridizable with a portion of the sequence set forth in SEQ ID NO:105.
The present invention also provides a polynucleotide probe comprising a nucleotide sequence specifically hybridizable with a portion of the nucleic acid molecule of claim 111.
The present invention also provides a polynucleotide probe comprising a nucleotide sequence specifically hybridizable with a portion of the nucleic acid molecule of claim 114.
The present invention also provides an antisense oligonucleotide up to 50 nucleotides in length comprising a nucleotide sequence which is 20 specifically hybridizable with the long mRNA transcript of human protein kinase C-a and which is not specifically hybridizable with the short mRNA transcript of human protein kinase C-ca.
The present invention also provides apolynucleotide probe comprising a nucleotide sequence specifically hybridizable to the long mRNA transcript of human protein kinase C-a.
The present invention also provides a method for detecting a gene coding for human protein kinase C-a in a sample comprising contacting the sample with a polynucleotide probe of claim 117 or claim 118 under conditions which allow for the formation of a polynucleotide duplex between the probe and said gene coding for protein kinase C-a; and detecting the presence or absence of a polynucleotide duplex whereby the presence of a polynicleotide duplex indicates the presence of said gene coding for human protein kinase C-a in said sample.
The present invention also provides a method for detecting the long mRNA transcript of human protein kinase C-a in a sample comprising contacting the sample with the polynucleotide probe of claim 123 under -6Dconditions which allow the formation of a polynucleotide duplex between the probe and the long mRNA transcript of human protein kinase C-ca and detecting the presence or absence of a polynucleotide duplex whereby the presence of a polynucleotide duplex indicates the presence of said long mRNA transcript of human protein kinase C-a in said sample.
The present invention also provides a method for modulating the expression of protein kinase C-a in a cell containing a protein kinase C-c gene comprising contacting the cell with an antisense oligonucleotide up to nucleotides in length, said antisense oligonucleotide comprising a nucleotide sequence specifically hybridizable with a portion of the sequence set forth in SEQ ID NO:105.
The present invention also provides a method for specifically modulating the expression of the long mRNA transcript of protein kinase C-ca in a cell containing a protein kinase C-ca gene comprising contacting the cell with an antisense oligonucleotide up to 50 nucleotides in length, said antisense oligonucleotide comprising a nucleotide sequence specifically hybridizable with a portion of the sequence set forth in SEQ ID NO:106.
The present invention also provides a method of treating an animal having a condition associated with protein kinase C-a comprising contacting 20 said animal with a therapeutically effective amount of an antisense oligonucleotide up to 50 nucleotides in length, said antisense oligonucleotide comprising a nucleotide sequence specifically hybridizable with a portion of the sequence set forth in SEQ ID NO:105.
The present invention also provides a method of treating an animal having a condition associated with expression of protein kinase C-a comprising contacting said animal with a therapeutically effective amount of an antisense oligonucleotide up to 50 nucleotides in length, said antisense oligonucleotide comprising a nucleotide sequence specifically hybridizable with a portion of the sequence set forth in SEQ ID NO:106.
7 BRIEF DESCRIPTION OF THE DRAWINGS Figure l(a) and 1(b) are graphical depictions of the effects on PKC expression of antisense oligonucleotides hybridizable with PKC-a. Oligonucleotides are arranged by PKC target region, 5' to 3'.
Figure 2 is a line graph showing dose-dependent reduction of PKC-a protein levels after oligonucleotide treatment of A549 cells. Y ISIS 4632; M ISIS 4649; ISIS 4636; A ISIS 4648.
Figure 3 is a bar graph showing reduction of PKC-a mRNNA after treatment of A549 cells with oligonucleotides.
Hatched bars represent the 8.5 kb transcript, plain bars represent the 4.0 kb transcript.
Figure 4 is a line graph showing the relationshiD between deoxy gap length and activity of chimeric oligonucleotides against PKC.
Figure 5 is a line graph showing dose response curves for chimeric oligonucleotides (all SEQ ID NO: 3) wi:h different deoxy gap lengths.
Figure 6 is a bar graph showing the effects of several 2'-O-methyl chimeric oligonucleotides of SEQ ID NO: 3 on PKC-a mRNA levels. Hatched bars represent the 8.5 kb 25 transcript, plain bars represent the 4.0 kb transcript.
Figure 7 is a bar graph and diagram showing the effects of several 2'-O-methyl and 2'-O-propyl chimeric oligonucleotides (6996, 7273) of SEQ ID NO: 3 on PKC-a mRNA levels. Hatched bars represent the 8.5 kb transcript, plain bars represent the 4.0 kb transcript.
Figure 8 is a bar graph and diagram showing the effects of additional 2'-O-methyl and 2 '-O-propyl chimeric oligonucleotides (7008, 7294) of SEQ ID NO: 3 on PKC-c mRNA levels. Hatched bars represent the 8.5 kb transcript, plain bars represent the 4.0 kb transcript.
a Figure 9 is a siet of bar graphs showing the ef fect of additional oligonuclectidep on P1CC-a MWXA levels- Figure 9A show oligonucleoLider. 6632, 6653 and 6665. Figure 9B showp oliganucleotide:a 3521 (for comparison) 7082, 7083 s and 7084. W~atched bars represent the 8.5 kcb transcript, plain bars represent the 4 .0 kb transcript- Figure 10 is a line graph showing anti-tumor acti.v1ty of ISIS 3521. Each dashed line represents tumnor volume in one animal created with control oligonucleotide; io each solid line represents rumor volume in one animal treated with ISIS 3521.
Figure 11 is~ a. bar graph showing effect of phosphorothioate oligonucleotides on PKC-ij expression in A549 cellS.
Figure 12 is a, nucleorcide sequence (SEQ ID NO: 104) of a portion, of the 31 uncranslared region of t-he human PKCa gene, beginning t the Bcl I aite near the I3 end of the previously known sequence and extending in the 3' direction. Newly determined sequences begin at nucleotide 56 and are uniderlined (SEQ ID NO -1.05) Bold sequences are unique to the long ITUNA transcript of PXCa (SEQ ID NO;1.06).
Figure 13 is a~ line graph showing a rnime course of P(Cci mRNA levels ini cells. (shown as percent of control) after creatmenit with oligonucleotide 7911 (SEQ ID NO: 1171.
Levels of both the s>iort and long TnRXA tr-anscripts are indicated. Levels of short mRNA~ transcript are represented by solid lines. Levels of long mnRNA transcript are represented by dotted lines. By 12 hours after treatmnent with ISIS 7911 (SEQ ID NO; 117), levels of both messages were reduced by over act- 1 9 SUMMARY OF THE INVENTION In accordance with the present invention, oligonucleotides are provided that are specifically hybridizable with DNA or RNA deriving from the gene that encodes PKC. The oligonucleotide comprises nucleotide units sufficient in identity and number to effect such specific hybridization. This relationship is commonly denominated as "antisense". In one preferred embodiment, the oligonucleotides are specifically hybridizable with the translation initiation codon of the gene, and preferably comprise a sequence CAT. In another preferred embodiment, the oligonucleotides are specifically hybridizable with the 5'-untranslated or 3'-untranslated regions of the gene. In yet another preferred embodiment, oligonucleotides are provided that are specifically hybridizable with DNA or mRNA encoding a particular PKC isozyme or a particular set of PKC isozymes. Such oligonucleotides may be conveniently and desirably presented in a pharmaceutically acceptable carrier.
20 In accordance with other preferred embodiments, the oligonucleotides comprise one or more chemical .9* modifications which convey some desired characteristic such as improved target affinity, cellular uptake or stability in the presence of cellular nucleases. Examples of modifications having such utility are 2'-O-alkyl and 2'fluoro sugar modifications and phosphorothioate backbone modifications.
Other aspects of the invention are directed to methods for modulating the expression of PKC or of a particular PKC isozyme or set of isozymes in cells or tissues. Additional aspects of the invention are directed to methods of detection in cells or tissues of the DNA or RNA that encodes PKC and specific detection in cells or tissues of RNA or DNA that encodes particular PKC isozymes.
Such methods comprise contacting cells or tissues suspected of containing said gene with oligonucleotides in accordance 10 with the invention in order to interfere with the effect of or to detect said RNA or DNA.
Other aspects of the invention are directed to methods for diagnostics and therapeutics of animals suspected of having a disease associated with PKC or one of its isozymes. Such methods comprise contacting the animal or cells or tissues or a bodily fluid from the animal with oligonucleotides in accordance with the invention in order to modulate the expression of PKC, to treat conditions associated with PKC, or to effect a diagnosis thereof.
This invention provides nucleic acid sequences that encode portions of the 3' untranslated region of human PKCa. Polynucleotide probes and methods of detecting PKCa are also provided. In some embodiments of the present 15 invention, nucleic acid sequences specific for a particular mRNA transcript of PKCa are provided, as well as polynucleotide probes and methods for specific detection of this transcript.
In accordance with other embodiments of the present 20 invention, antisense oligonucleotides are provided that are specifically hybridizable with nucleic acids encoding PKCa.
In still other embodiments, antisense oligonucleotides are provided which are specifically hybridizable with a particular mRNA transcript of PKCa. Such oligonucleotides 25 may be conveniently and desirably presented in a pharmaceutically acceptable carrier.
In accordance with still other aspects of the invention are provided methods for modulating the expression of PKCa or of a particular PKCa mRNA transcript in cells. Additional aspects of the invention are directed to methods of detection in cells of nucleic acids that encode PKCa and specific detection in cells of nucleic acids that encode particular PKCa transcripts. Such methods comprise contacting the cells with oligonucleotides in accordance with the invention in order to interfere with the effect of or to detect said nucleic acid.
11 In still other embodiments of the invention are provided methods for treating animals having a disease associated with expression of PKCa or one of its transcripts. Such methods comprise contacting the animal with a therapeutically effective amount of oligonucleotides in accordance with the invention in order to modulate the expression of PKCa, to treat conditions associated with PKCa, or to effect a diagnosis thereof.
DETAILED DESCRIPTION OF THE INVENTION Antisense oligonucleotides are now accepted as therapeutic agents having promise for the treatment of many human diseases. Oligonucleotides specifically bind (hybridize) to the complementary sequence of DNA, pre-mRNA or mature mRNA, as defined by Watson-Crick base pairing, 15 interfering with the flow of genetic information from DNA to protein. The properties of antisense oligonucleotides which make them specific for their target sequence also make them extraordinarily versatile. Because antisense oligonucleotides are long chains of monomeric units, they may be readily synthesized for any target RNA sequence.
Numerous recent studies have documented the utility cf antisense oligonucleotides as biochemical tools for studying target proteins (Rothenberg et al., J. Natl.
Cancer Inst., 81:1539-1544 (1989); Zon, Pharmaceutical Res., 5:539-549 (1988). Because of recent advances in oligonucleotide chemistry and synthesis of oligonucleotides which exhibit enhanced cell uptake, target binding affinity and nuclease resistance, it is now possible to consider the use of antisense oligonucleotides as a novel form of therapeutics. For example, antisense oligonucleotides targeted to c-myb have been used to completely eliminate myeloid leukemia cells from bone marrow derived from patients with acute myelogenous leukemia. Gewirtz and Calabretta, U.S. Patent 5,098,890. An antisense oligonucleotide has been shown to have clinical efficacy in 12 humans for treatment of cytomegalovirus retinitis infections.
Antisense oligonucleotides offer an ideal solution to the problems encountered in prior art approaches to the treatment of conditions associated with PKC. They can be designed to selectively inhibit a given isozyme or particular set of isozymes, or to inhibit all members of a given family of isozymes.
Current agents which modulate the activity or metabolism of protein kinase C exhibit many unacceptable side effects due to their lack of specificity, or they exhibit only limited effectiveness in inhibiting the enzyme. The instant invention circumvents problems encountered by prior workers by modulating the production 15 of the enzyme, rather than inhibiting the enzyme directly, to achieve the therapeutic effect. In the instant invention, the oligonucleotide is designed to hybridize directly to mRNA or to a gene, ultimately modulating the amount of PKC protein made from the gene. "Hybridization," 20 in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand, to form a double-stranded duplex. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are *examples of complementary bases which are known to form two hydrogen bonds between them. "Specifically hybridizable" and "substantially complementary" are terms which indicate a sufficient degree of complementarity to avoid nonspecific binding of the oligonucleotide (or polynucleotide probe) to non-target sequences under conditions in which specific binding is desired, under physiological conditions in the case of in vivo assays and therapeutic treatment, or, in the case of in vitro assays, under conditions in which the assays are conducted. It is understood that an oligonucleotide or polynucleotide probe 13 need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.
The relationship between an oligonucleotide and its complementary (or "target") nucleic acid is commonly denoted as "antisense." It is preferred to target specific genes for antisense attack. It has been discovered that the genes coding for PKC a, S, 7, 6, e, and 7 are particularly useful for this approach. Inhibition of PKC expression is expected to be useful for the treatment of diseases, particularly hyperproliferative and inflammatory disorders.
However, "modulation" in the context of this invention means either an increase or decrease (stimulation or inhibition) of PKC expression.
15 In the context of this invention, the term "oligonucleotide" refers to a polynucleotide formed from naturally occurring nucleobases and pentofuranosyl (sugar) groups joined by native phosphodiester bonds. This term effectively refers to naturally occurring species or 20 synthetic species formed from naturally occurring subunits or their close homologs.
The term "oligonucleotide" may also refer to moieties which function similarly to naturally occurring oligonucleotides but which have non-naturally occurring portions. Thus, oligonucleotides may have altered sugar Smoieties, nucleobases or inter-sugar ("backbone") linkages.
Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake, enhanced target binding affinity and increased stability in the presence of nucleases.
Specific examples of some preferred oligonucleotides envisioned for this invention are those which contain intersugar backbone linkages such as phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those 14 with CH 2
-NH-O-CH
2
CH
2
-N(CH
3
CH
2
-O-N(CH
3
)-CH
2
CH,-
N(CH
3
)-N(CH
3
)-CH
2 and O-N(CH) -CH 2
-CH
2 backbones (where phosphodiester is Phosphorothioates are also most preferred. Also preferred are oligonucleotides having morpholino backbone structures. Summerton, J.E. and Weller, U.S. Patent 5,034,506. In other preferred embodiments, such as the peptide nucleic acid (PNA referred to by some as "protein nucleic acid") backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone wherein nucleosidic bases are bound directly or indirectly to aza nitrogen atoms or methylene groups in the polyamide backbone, see, P.E. Nielsen, M. Egholm, R.H. Berg, O. Buchardt, .Science 1991, 254, 1497 and United States Patent 15 Application Serial No. 08/054,363, filed April 26, 1993 and incorporated herein by reference. In accordance with other preferred embodiments, the phosphodiester bonds are substituted with structures which are chiral and enantiomerically specific. Persons of ordinary skill in 20 the art will be able to select other linkages for use in practice of the invention.
Oligonucleotides may also include species which include at least one modified nucleobase. Thus, purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the pentofuranosyl portion of the nucleotide subunits may also be effected, as long as the essential tenets of this invention are adhered to. Examples of such modifications are 2'-O-alkyl- and 2'-halogen-substituted nucleotides.
Some specific examples of modifications at the 2' position of sugar moieties which are useful in the present invention are OH, SH, SCH 3 F, OCN, O(CH 2 )nNH 2 or O(CH 2
),CH
3 where n is from 1 to about 10; C 1 to lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3
OCF
3 or N-alkyl; or N-alkenyl;
SOCH
3
SOCH
3 ONO2; NO 2
N
3 NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; 15 a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. One or more pentofuranosyl groups may be replaced by another sugar, by a sugar mimic such as cyclobutyl or by another moiety which takes the place of the sugar.
Chimeric or "gapped" oligonucleotides are also preferred embodiments of the invention. These oligonucleotides contain two or more chemically distinct regions, each comprising at least one nucleotide.
Typically, one or more region comprises modified nucleotides that confer one or more beneficial properties, 15 for example, increased nuclease resistance, increased uptake into cells or increased binding affinity for the RNA target. One or more unmodified or differently modified regions retain the ability to direct Rnase H cleavage.
Chimeric oligonucleotides are disclosed in PCT application 20 US92/11339 which is assigned to the assignee of the instant application and which is incorporated by reference herein in its entirety. Examples of chimeric oligonucleotides which are presently preferred are 2'-O-methyl or propyl oligonucleotides having a "deoxy gap" region of 2'deoxynucleotides. Usually this deoxy gap region is located between the two 2'-alkyl regions. In these preferred embodiments, the internucleotide (backbone) linkages may be uniformly phosphorothioate or some combination of phosphorothioate and phosphodiester linkages.
All such oligonucleotides are best described as being functionally interchangeable with natural oligonucleotides (or synthesized oligonucleotides along natural lines), but having one or more differences from natural structure. All such oligonucleotides are comprehended by this invention so long as they function effectively to hybridize with the PKC RNA.
16 The oligonucleotides in accordance with this invention preferably comprise from about 5 to about nucleotide units. It is more preferred that such oligonucleotides comprise from about 8 to 30 nucleotide units, and still more preferred to have from about 12 to nucleotide units. As will be appreciated, a nucleotide unit is a base-sugar combination (or a combination of analogous structures) suitably bound to an adjacent nucleotide unit through phosphodiester or other bonds forming a backbone structure.
The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of the routineer. It is also well known to use similar techniques to prepare other oligonucleotides such as phosphorothioates S 20 or alkylated derivatives. Other modified and substituted oligomers can be similarly synthesized.
In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the coding region, which contains 25 information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the region, the 3 '-untranslated region, the 5' cap region and intron/exon junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the coding ribonucleotides. In preferred embodiments, the oligonucleotide is specifically hybridizable with a transcription initiation site, a translation initiation site, a 5' cap region, an intron/exon junction, coding 17 sequences or sequences in the or 3 '-untranslated region.
The oligonucleotides of this invention are designed to be hybridizable with the PKC gene or with messenger RNA derived from the PKC gene. Such hybridization, when accomplished, interferes with the normal roles of the messenger RNA to cause a modulation of its function in the cell. The functions of messenger
RNA
to be interfered with may include all vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and possibly even independent catalytic activity which may be engaged in by the RNA. The overall effect of such 15 interference with the RNA function is to modulate expression of the PKC gene.
The oligonucleotides of this invention can be used in diagnostics, therapeutics, prophylaxis, and as research reagents and kits. Since the oligonucleotides of this 20 invention hybridize to the PKC gene and its mRNA, sandwich and other assays can easily be constructed to exploit this fact. Furthermore, since the oligonucleotides of this invention hybridize specifically to particular isozymes of the PKC mRNA, such assays can be devised for screening of cells and tissues for particular PKC isozymes. Such assays can be utilized for diagnosis of diseases associated with various PKC forms. Provision of means for detecting hybridization of oligonucleotide with the PKC gene can routinely be accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of PKC may also be prepared.
For therapeutic or prophylactic treatment, oligonucleotides are administered in accordance with this invention. Oligonucleotides may be formulated in a pharmaceutical composition, which may include carriers, thickeners, diluents, buffers, preservatives, surface is8 active agents 4nd the like in addition to the olisonucleotide. Phazmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, ant ±inflammatory agents. anesthetics, and the like in addit:Lon to ol goaucleat ides The pharmaceutical composition may he administered in a number of ways depending on whether local or systewic treatment is dsired, and on rhe area to be treated.
Administrti.on may be done topically (including ophthalmically, vaginally, rectally, intranasally), orally.
by inhalation. or parenterally, for example by intravenous drip or subcutaneous, intraperitoneal or intramuscular injectionl- Formnulations for zopical administration may include creams, gels, drops. suppositories, sprays, li~quids and powders- Conventi.onal pharmaceutical 7....carriers, aqueous. powder or oily bases. thickener-B and the like may be necessary or desirable. Coated condoms may also be useful.
Compositions for oral administration include powders or granules, suspensions or solutions in water or nonaqueous mediia, capaules, sachets, or tablets. Thickeners, flavorings, diluerits, emulsifier5, dispersing aid.s or binders may be desirable.
Formulations f.or pareateral administration may include sterile aqueous solutions which may also contain buffers, diluentu Emid other suitable additi.ves.
Dosing is dependent on severity and respon.9iirenes of the coudicion to lae treated, but will normally be one or more doses per clay, w~ith course of treatment lasting from several days to several months or until a cure is effected or a dimiinution ot disease state is achieved. Personls of ordinary skill can easily determrinle optimum dosages, dosing methodologies and repetition rates.
The present invention also provides a nucleic acid molecule having a sequence which encodes the 32untranslated region of human PKCa is provided (Figure 12J 19 This sequence w&3 determined fromn cDNA clones Prepared from huan As49 cells, beg:Lnniflg with a clone overlapping the 3' -moor and of the previously published PKCea sequence [Flukenzeller et ?Jucl. Acids Reg. 18:2I.5 3 (1990); Genbazlk accession nuiiWber X524'/5] and extending in the 3' direction. A polyadeny Vlation site which was reached after 1080 nucleorides (rucleoride 1136 in Figure 12); has been identifi-ed as the 3, end of Lhe short (4 kb) mRYA transcript of PKC&- An' additional 676 aucleotides of sequen~ce in the 3' direction were determined, which sequence ip unique to the long (8kb) WMPA transcript of Mix-. The nucleic acid molecule of the present inlvenftionl may preferrably be comprised of deoxyribonucleic acids and may be double-stranded in some aspects of the prepear isinvention. Also in accordance with the present in-tention, 2 said nucleic acid molecules are isolated. ,Isolated,, as the term is used herein, in mfeant to refer to molecules which have been purified or synthesized so as to be subatantially hornogenouE. The term does nor exclude the porssibillry thar- certain impurities may be present in the composicion, but is, instead, meant to refer to the absence of non-relevant nucleic acid sequences.
in accordance with the present invention polynucleotide probe~s specifically hybridizable, to a portion of the 3' unizrans].aced region of the human PKCa gene are provided. ,?oiynucleocide probes rpecitically hybridizable to a porcion of r-he long niRWA tranS;cript_ of PXCa are al.so provided. Such probes may bae used for diagnoscic or research purposes to detect or quanritare the expreasion of PKC&. Probe4 may be used to specitically detect or quantitate the long transcript of PKCa. Said polynucleotide probes may range in length from about 5 to about 5o nucleocide units. in more preferred embodiments of the present invention the probes may be from abot 8 to 3S about 30 nuclaotide unitN in length. Ideally. said probes range in length from about 12 to about 25 nucleotide units.
It is recognized thZr since polynucleotide probes of the 20 present invention ideally do not exceed 50 nucleotides in length, said probes may specifically hybridize to only a portion of the targeted sequence. The portion of the PKCa sequence to be targeted can be identified by one skilled in the art. Most suitably, a target sequence is chosen which is unique, thereby decreasing background noise attributable to hybridization by the probe other than to the target. By way of example, one skilled in the art would be unlikely to select a repeating sequence of adenine nucleotide units as this is a common sequence occurring in many genes. The practitioner might choose to perform a search and comparison of sequences found in a sequence depository such as Genbank in order to identify and design a useful probe.
S• Such methods are conventionally used to identify unique 15 sequences. These unique sequences, when used as probes, need not necessarily be crucial to the regulation of the expression of PKCa.
The following examples illustrate the present invention and are not intended to limit the same.
EXAMPLES
Example 1 Oligonucleotide synthesis: Unmodified DNA oligonucleotides were synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with 25 oxidation by iodine. S-cyanoethyldiisopropylphosphoramidites were purchased from Applied Biosystems (Foster City, CA). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step was increased to 68 secondd and was followed by the capping step.
2'-O-methyl phosphorothioate oligonucleotides were synthesized according to the procedures set forth above substituting 2'-O-methyl -cyanoethyldiisopropyl phosphoramidites (Chemgenes, Needham, MA) for standard 21 phosphoramidites and increasing the wait cycle after the pulse delivery of tetrazole and base to 360 seconds.
Similarly, 2'-O-propyl phosphorothioate oligonucleotides may be prepared by slight modifications of this procedure.
After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 550C for 18 hours, the oligonucleotides were purified by precipitation twice out of 0.5 M NaCI with 2.5 volumes ethanol. Analytical gel electrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mM Tris-borate buffer, Ph The oligonucleotides tested are presented in Table 1. Sequence data are from the cDNA sequence published by Finkenzeller et al., Nucl. Acids Res. 18:2183 (1990); 15 Genbank accession number X52479. The sequence numbers given under the oligonucleotides are relative to the first residue to be sequenced on the cDNA, which is 28 residues upstream of the ATG start codon.
Table 1 OLIGONUCLEOTIDES TARGETED TO HUMAN PKC-a SEQ Sequence Target ISIS
ID
1 CCC CAA CCA CCT CTT GCT CC 5' 3520 19 1 Untranslated S 25 2 GTT CTC GCT GGT GAG TTT CA 3' 3521 2063 2044 Untranslated 3 AAA ACG TCA GCC ATG GTC CC Translation 3522 41 22 init. codon 4 GGA TTC ACT TCC ACT GCG GG 3' 3526 2109 2090 Untranslated GAG ACC CTG AAC AGT TGA TC 3' 3527 221 1 2192 Untranslated 6 CCC GGG AAA ACG TCA GCC AT Translation 3674 47 28 init codon 7 CTG CCT CAG CGC CCC TTT GC Internal 3682 110 91 (C1) domain 22 8 AGT CGG TGC AGT GGC TGG AG 193 174 9 10 11 12 13 14 15 15 16 17 18 GCA GAG 480 GGG CTG 2080 CAC TGC 2098 AGC CGT 2137 ATT TTC 2168 AAG AGA 2217 GAT AAT 2235 ATG GGG 2027 GTC AGC 36 CGC CGT *ft ft..
ft
GC]
GGC
GGG
GGC
AGG
GAG
GTT
TGC
CAT
GGA
3GA
"AG
r GGC
SAGC
GAG
SCTT
CCT
ACC
CTT
ACA
GGT
GTC
GGC
ACA
GAC ATT GA 461 TGT TTG TT 2061 GGC TGG GG 2079 AAA ATT TT 2118 CCA TAT GG 2149 CTG AAC AG 2198 GGT TGT AA 2216 AAC TGG GG 2008 CCC CCC CC 17 GTT GCC CG 44 TGC CCG GC 1624 CAA GCC GT 2132 Internal (Cl) domain Internal (Cl) domain 3' Untranslated 3' Untranslated 3' Untranslated 3' Untranslated 3' Untranslated 3' Untranslated Internal (C3) domain Translation init. codon Internal (VI) domain Internal (C3) domain 3' Untranslated 3686 3687 3695 3875 3878 3879 3884 3885 3886 3890 3891 3892 3947 19 20 TCA AAT 1643 TGG AAT 2151 Example 2 Cell culture and treatment with phorbol esters and oligonucleotides targeted to PKC-a: PKC protein half-lives have been reported to vary from 6.7 hours to over 24 hours [Young et al., Biochem.
J.
244:775-779 (1987); Ballester et al., J. Biol. Chem.
260:15194-15199 (1985)]. These long half-lives make inhibiting steady-state levels of PKC-a an unwieldy approach when screening antisense oligonucleotides, due to the long incubation times which would be required. We have therefore made use of the ability of phorbol esters to reversibly lower intracellular levels of PKC. Treatment of 23 cells with phorbol esters causes an initial activation of kinase activity, followed by a down-regulation of PKC. For PKC-a this down-regulation has been shown to be a direct consequence of an increased rate of proteolysis of the kinase with no apparent change in synthetic rate.
We determined that in human lung carcinoma (A549) cells, treatment with the phorbol ester 12,1 3 -dibutyrate (PDBu), using a modification of the method of Krug et al., [Krug et al., J. Biol. Chem. 262:11852-11856 (1987)] lowered cellular levels of PKC-a, without affecting PKC-a mRNA levels, and that this effect was reversible. The basis of the assay to screen for potency of oligonucleotides targeting PKC-a is to initially lower
PKC-
Sprotein levels by chronic treatment with PDBu, remove 15 PDBu by extensively washing the cells (hence allowing the cells to synthesize fresh PKC-a protein), and incubate the cells with oligonucleotides intended to inhibit the *resynthesis of new PKC-a protein.
Procedure: A549 cells (obtained from the American 20 Type Culture Collection, Bethesda MD) were grown to confluence in 6-well plates (Falcon Labware, Lincoln Park, .NJ) in Dulbecco's modified Eagle's medium (DME) containing 1 g glucose/liter and 10% fetal calf serum (FCS, Irvine Scientific, Santa Ana,
CA).
Cells were treated with 500 nM PDBu (Sigma Chem SCo., St. Louis, MO) for 12-16 hours (overnight) Cells were then washed three times in DME at 37oC, and 1 ml DMA containing 20 Al DOTMA (Lipofectin reagent, BRL, Bethesda, MD) was added. Oligonucleotides were added to a concentration of pM and the cells were incubated for a further 4 hours at 371C.
Cells were washed once in 3 ml DME containing 0.1 mg/ml BSA and a further 2 ml DME containing 0.1 mg/ml
BSA
was added. Oligonucleotides (2 pM) were added and the cells were incubated at 37 0 C for 24 hours.
Cells were washed three times in phosphate-buffered saline (PBS) and cellular proteins were extracted in 120 Al 24 sample buffer (60 mM Tris pH 6.8, 2% SDS, 10% glycerol, mM dithiothreitol) and boiled for 5 minutes. Intracellular levels of PKC-a protein were determined by immunoblotting.
Example 3 Immunoblot assay for PKC expression: Cell extracts were electrophoresed on 10% SDS-PAGE mini-gels. The resolved proteins were transferred to Immobilon-P membrane (Millipore, Bedford MA) by electrophoretic transfer and the membrane was blocked for minutes in TBS (Tris-HCl pH 7.4, 150 mM NaCl) containing 5% nonfat milk. The membrane was then incubated for 16 hours at 40C with monoclonal antibodies raised against PKCa (UBI, Lake Placid NY) diluted to 0.2 yg/ml in TBS containing 0.2% nonfat milk. This was followed by three washes in TBS plus 0.2% nonfat milk. The membrane was then incubated for one hour with 125 I-labelled goat anti-mouse secondary antibody (ICN Radiochemicals, Irvine CA).
Membranes were then washed extensively in TBS plus 0.2% nonfat milk. Bands were visualized and quantitated using a Phosphorimager (Molecular Dynamics, Sunnyvale, CA). PKC-a appears as a single band with a molecular weight of 80 kD.
Each oligonucleotide was tested three times, in triplicate, and the results of the experiments were normalized against percentage of protein present as compared to cells which were not treated with oligonucleotide (Figures la and Ib). The five most effective oligonucleotides target the AUG start codon and regions slightly upstream and downstream from it (Sequence Nos. 1, 3, 17, 7, The next most effective oligonucleotides are targeted toward the 3' untranslated region of the RNA (oligos 2, 5, 14).
25 Example 4 Other isozymes of PKC: Results with oligonucleotides targeting human PKC-a demonstrated that the most effective target sequences were those surrounding the translation initiation codon and the 3' untranslated region. It is believed that these sequences will also be effective targets for oligonucleotides directed against other isozymes of PKC.
Antisense oligonucleotides which are likely to be effective inhibitors of PKC are identified below. These oligonucleotides are synthesized as in Example 1, and can be screened as in Examples 2 and 3, using appropriate antibodies where available. Alternatively, a reporter gene assay system can be established, transiently co-expressing the desired isozyme of PKC with luciferase under the 15 influence of the TPA-responsive enhancer or other suitable promoter. PKC expression is then assayed by measuring luciferase activity using standard procedures. Luciferase is extracted from cells by lysis with the detergent Triton X-100, as described by Greenberg, in Current 20 Protocols in Molecular Biology, Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.A. Smith, J.G. Seidman and K.
Strahl, eds.), John Wiley and Sons, NY (1987). A Dynatech S"ML1000 luminometer is used to measure peak luminescence upon addition of luciferin (Sigma) to 625 pM.
PKC-f, types I and II Sequence data are from Kubo et al., FEBS Lett. 223: 138-142 (1987); Genbank accession numbers X06318, M27545, X07109. Sequences are numbered from the first 5' base sequenced on the cDNA. PKC-f types I and II are the result of alternative mRNA splicing of a single gene product.
This results in proteins with identical amino termini end of. the mRNA); however, there is sequence divergence in the carboxy termini end of the mRNA). The following oligonucleotides, targeted to the translation initiation codon, are expected to modulate expression of both PKC-G types I and II: 26 TABLE 2 OLIGONUCLEOTIDES TARGETED TO PKC-9 TYPES I AND 11 SEQ ID Sequence Target 21 CAT CTT GCG CGC GGG GAG CC Translation init.
139 120 22 TGC GCG CGG GGA GCC GGA GC 134 115 23 CGA GAG GTG CCG GCC CCG GG 113 94 24 CTC TCC TCG CCC TCG CTC GG 183 164 The following antisense oligonucleotjdes are targeted to the 3 1 -untranslated region of PKC-S type
I:
TABLE 3 15 OLIGONUCLEOTIDES TARGETED TO PKC-IZ TYPE
I
SEQ.In Sequence Tarqret TGG AGT TTG CAT TCA CCT AC 3' Untranslated 2168 2149 26 AAA CGC CTC TAA GAC AA CT 11 1 5- 5* S 5 *5C*
S
20 27 28 29 2285 2266 GCC AGC ATG TGC ACC GTG AA 2250 2231 ACA CCC CAG GCT CAA CGA TG 2186 2167 CCG AAG CTT ACT CAC AAT TT 2569 2550 The following antisense oligonuclectides are targeted to the 3 '-untranslated region of PI(C-9 Type II: 27 TABLE 4 OLIGONUCLEOTIDES TARGETED TO PKC- TYPE II SEQ. ID Sequence Target 31 32 33 ACT TAG CTC TTG ACT TCG GG 2160 2141 ATG CTG CGG AAA ATA AAT TG 2420 2401 ATT TTA TTT TGA GCA TGT TC 2663 2644 TTT GGG GAT GAG GGT GAG CA 2843 2824 CCC ATT CCC ACA GGC CTG AG 3137 3118 3' Untranslated II r I, ii ro r *r~ PKC-y: 15 Sequence data are from Coussens et al., Science 233:859-866 (1986); Genbank accession number M13977.
Sequences are numbered from the first 5' base n'en~e~ad in the cDNA. The full sequence is not available: the extreme 3' end of the open reading frame and the 3' untranslated region are missing. Consequently these regions are not presently available as antisense targets.
TABLE OLIGONUCLEOTIDES TARGETED TO PKC-y
SEQ.ID
Sequence Target 35 36 37 CGG AGC GCG CCA GGC AGG GA 51 32 CCT TTT CCC AGA CCA GCC AT 215 196 GGC CCC AGA AAC GTA GCA GG 195 176 GGA TCC TGC CTT TCT TGG GG 170 151 CAG CCA TGG CCC CAG AAA CG 202 183 5' Untranslated Translation init.
5' of start codon 5' Untranslated Translation init.
28
PKC-
1 7: Sequence data for PKC-n are from Bacher and colleagues [Bacher et al., Mol. Cell. Biol. 11:126-133 (1991)]; Genbank accession number M55284. They assign their isozyme the name PKC-L; however the sequence is almost identical to that of mouse PKC-n, so the latter nomenclature is used here for consistency. Sequences are numbered from the first 5' base sequenced in the cDNA.
TABLE 6 0 OLIGONUCLEOTIDES TARGETED TO PKC-n
SEQ.ID
Sequence Target CGA CAT GCC GGC 172 1 r r r r r r r r r rr r r r r r r cr r 41 15 42 43 20 44 46 25 47 48 49 CAG ACG 176 GCC TGC 138 ACA GGT 86 GTC CCG 111 CCT CAC 221 ATT GAA 193 TCT CAC 2046 TTC CTT 2067 TTC CAT 2353 AGG CTG 2300
ACA
TTC
GCA
TCT
CGA
CTT
TCC
TGG
CCT
ATG
STGC
GCA
GGA
CAG
TGC
CAT
CCA
GTT
TCG
CTG
GCC
CGG
GCG
GTC
GCC
GGA
GGT
TAA
CTC
ACA
GGA
GCT GC 153 CGC CG 157 GGA GA 119 GAG GC 67 AGC CC 92 CCC TC 202 GCC AG 174 GGC TA 2027 GTG CC 2048 GAG TT 2336 AGG TC 2281 CTG GG 2287 Translation init.
3' Untranslated I, 11i 3' Untranslated S It GTT CTA 2306 AGG CTG ATG 29 Example 5 Dose response of oligonucleotide effects on PKC-a protein synthesis: A series of phosphorothioate, fully 2 '-O-methyl oligonucleotides having SEQ ID NO: 1, 2, 3 and 5 were synthesized. A549 cells were treated with 500 nM PDBu for 18 hours to downregulate PKC-a synthesis, washed to remove PDBu and then treated with oligonucleotide and DOTMA/DOPE cationic liposomes. Medium was replaced after four hours and the cells were allowed to recover for another 20 hours.
Proteins were extracted and PKC-a protein levels were determined by immunoblotting as described in Example 3.
Results were quantified with a phosphorimager (Molecular Dynamics, Sunnyvale CA) and are shown in Figure 2 expressed as percent of control (saline treatment). ISIS 4649 (SEQ ID 15 NO: 3; squares) reduced PKC-a protein levels by 85-90% at 500 nM and had an IC50 of approximately 260 nM.
Example 6 Effect of antisense oligonucleotides on PKC-a mRNA levels: A549 cells were treated with phosphorothioate 20 oligonucleotides at 500 nM for four hours in the presence of the cationic lipids DOTMA/DOPE, washed and allowed to recover for an additional 20 hours. Total RNA was extracted and 20Ag of each was resolved on 1.2% gels and transferred to nylon membranes. These blots were probed with a "P 25 radiolabeled PKC-a cDNA probe and then stripped and reprobed with a radiolabeled G3PDH probe to confirm equal RNA loading. Each oligonucleotide (3520, 3521, 3522 and 3527) was used in duplicate. The two major PKC-a transcripts (8.5 kb and 4.0 kb) were examined and quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale CA). Results are shown in Figure 3.
Oligoncleotides 3521 (SEQ ID NO: 3522 (SEQ ID NO: 3) and 3527 (SEQ ID NO: 5) gave better than 50% reduction of PKC-a mRNA levels. Oligonucleotides 3521 and 3527 gave approximately 80% reduction of the smaller transcript and over 90% reduction of the larger transcript.
30 Example 7 Chimeric (deoxy gapped) 2 '-O-methyl oligonucleotides: Oligonucleotides 3521 (SEQ ID NO: 3522 (SEQ ID NO: 3) and 3527 (SEQ ID NO: 5) were chosen for further study and modification. Oligonucleotides having these sequences were synthesized as uniformly phosphorothioate chimeric oligonucleotides having a centered deoxy gap of various lengths flanked by 2'-O-methylated regions. These oligonucleotides (500 nM concentration) were tested for effects on PKC-a mRNA levels by Northern blot analysis.
Results are shown in Figure 4. Deoxy gaps of eight nucleotides or more gave maximal reduction of PKC-a mRNA levels (both transcripts) in all cases. The oligonucleotide having SEQ ID NO: 3 reduced PKC-a mRNA by approximately 83% with a deoxy gap length of four nucleotides, and gave nearly complete reduction of PKC-a mRNA with a deoxy gap length of six or more.
Dose-response curves for these oligonucleotides are shown in Figure 5. The 2'-O-methyl chimeric 20 oligonucleotides with four- or six-nucleotide deoxy gaps have an IC50 for PKC-a mRNA reduction (concentration of oligonucleotide needed to give a 50% reduction in PKC-a mRNA levels) of 200-250 nM, as did the full-deoxy oligonucleotide (all are phosphorothioates throughout). The 25 2'-O-methyl chimeric oligonucleotide with an 8-nucleotide deoxy gap had an IC50 of approximately 85 nM.
Several variations of this chimeric oligonucleotide (SEQ. ID NO: 3) were compared for ability to lower PKC-a mRNA levels. These oligonucleotides are shown in Table 7.
31 Table 7 Chimeric 2 1 -O-methyl/deoxy P=S oligonucleotides bold= 2'-O-methyl; s= P=S linkage, o= P=O linkage OLIGO SEQUENCE SEQ ID NO: 3522 AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC 3 5352 AsAsAsAsCsGsTsCsAsGsCsCsAsTsGGsTsCsCsC 3 6996 AoAoAoAoCOGsTsCsAsGsCsCsAsTsGoGoToCoCaC 3 7008 AsAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCsC 3 7024 ABAoAoAoCoGsToCsAoGsCoCsAsTsGoGoToCoCSC 3 Effects of these oligonucleotides on PKC-a mRNA levels is shown in Figure 6. Oligonucleotides 7008, 3522 and 5352 show reduction of PKC-a mPNA, with 5352 being most active.
A series of 2'-O-propyl chimeric oligonucleotides was synthesized having SEQ ID NO: 3. These oligonucleotides are shown in Table 8.
Table 8 Chimeric 2'-O-propyl/deoxy P=S oligonucleotides bold= 2'-O-propyl; s= P=S linkage, o= P=O linkage OLIGO SEQUENCE SEQ ID NO: 7199 AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCs C 3 7273 AoAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCoC 3 7294 AsAoAoAocoGsTsCsAsGsCsCsAsTsGoGoToCoCsC 3 7295 AsAoAoAoCoGsToCsAoGsCoCsAsTsGoGoToCoCsC 3 These 2'-O-propyl chimeric oligonucleotides were compared to the 2'-O-methyl chimeric oligonucleotides.
Oligonucleotides 7273 and 7294 were more active than their 2'-O-methyl counterparts at lowering PKC-a mRNA levels.
This is shown in Figures 7 and 8.
Example 8 Additional oligonucleotides which decrease PKC-a mRNA: Additional phosphorothioate oligonucleotides targeted to the human PKC-a 3' untranslated region were 32 designed and synthesized. These sequences are shown in Table 9.
Table 9 Chimeric 2'-0-propyl/deoxy P=S oligonucleotides targeted to PKC-a 3'-UTR bold= 2 '-O-propyl; s= P=S linkage, o= P=O linkage OLIGO SEQUENCE SEQ ID NO: 6632 TsTsCs TsCsGs CsTsGs GsTsGs AsGsTs TsTsC 52 6653 TsTsCs TsCsGs CsTsGs GsTsGs AsGsTs TsTsC 52 6665 ToToCo TsCsGs CsTsGs GsTsGs AsGsTo ToToC 52 7082 TsCsTs CsGsCs TsGsGs TsGsAs GsTsTs TsC 53 7083 TsCsTs CsGsCs TsGsGs TsGsAs GsTsTs TsC 53 7084 ToCoTo CsGsCs TsGsGs TsGsAs GsToTo ToC 53 As shown in Figure 9, oligonucleotides 6632, 6653, 7082 and 7083 are most active in reducing PKC-a mRNA levels.
U.
Example 9 Culture of human A549 lung tumor cells: *The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (Bethesda MD). Cells were grown in Dulbecco's Modified 20 Eagle's Medium (Irvine Scientific, Irvine CA) containing 1 ngm glucose/liter and 10% fetal calf serum (Irvine Scientific). Cells were trypsinized and washed and *resuspended in the same medium for introduction into mice.
Example 10 Effect of ISIS 3521 on the growth of human A549 tumor cells in nude mice: 200 g1 of A549 cells (5 x 106 cells) were implanted subcutaneously in the inner thigh of nude mice.
ISIS 3521, a phosphorothioate oligonucleotide with Sequence ID NO 2 was administered twice weekly for four weeks, beginning one week following tumor cell inoculation.
Oligonucleotides were formulated with cationic lipids (DMRIE/DOPE) and given subcutaneously in the vicinity of the tumor. Oligonucleotide dosage was 5 mg/kg with 60 mg/kg cationic lipid. Tumor size was recorded weekly.
33 As shown i~n Figure 10, tumor growth was almost completely inhibited iLn two of the three mice, and reduced compared to control in the third mouse- Thip inhibitrion of tumor growth by ISIS 3521 is ptar4isticallY Significant. The control o:Uigonucleotj,:e (ISIS 1082) is a 21-wier phosphorothicate oJligonucleotide without sigsllficaflt sequence homology to -the P1CC n'R)A carget.
Admistratiofl of oligonucleotides to mice whose tumors had already reached detectabl Size had no discernable effect on aubsequent tumor grow~th.
Example 11 E~ffect of axuti.sene oligonuclaorides on growth of human X4DA-IMS231 tumors in nude Nice; MDA-MB231 human breast carcinoma cells are obtained from the American Type Cu~lzure Colilect-ion 125 (Bethesda, MD) Serially tranigplanred MDA-M0231 cumors are established subcutaneously in nude mice. Beginning two weeks later, o1l.gonucleot-idas 3521 and 3527, a phosphorothi.oate olicionuclearide having Sequence ID NO. in saline, are administered intravenously daily for 1~4 days at dosages between 2 rri/kg and 25 mg/kg. Cointrol oligonucleotide ISIS 1082 ir. also administered at these and a saline ccontrol is also given- Tumor growth rates are monitoredl for the two-week period of 5.95aoligonucleotide adminitracion.
34 Ficample 12 Effeet3 of antiseflee oliqglluclectide Isis 4155 all endogez~ua Pmc-a expreasion in hairless mice: In order to~ study oligonucleoride effects on endogenous PKC mRNA levels in normal ani.mals, it was necessary to employ an oligoziucleotide complementary to the murine PICC-a. ISIS 4189 to a 20-mer phospkhorothicate oliqonucleot-de rargecad to the AUG codon of tuouse PKC-a.
This region is without homology to the human PKC! sequence and Lhe oligonucleotide has no effect an expression of PKCa in human calls. ISIS 4189 has an IC50 of 200 nXM for mnRNA reduction in C127 mouse breast epithel~ial cells. ISIS 1*189 :L saline was adminiitered intraperitoneally to hairless m~ice at concentrations of 1. 10 or 100 mg/kg body weight.
Injections were givent daily for seven days. Tissues from liver, kidney, spleenL, lung and skin were removed and ?PCC-cx TTRNA and protein levels were determined. iHisropathologica.
examination wao also performed on liver, kidney and lung samples. ISIS 4189 at: 100 ing/kg Inh~bited andogenouo PYC-a mRNA'levels in the mcouqe liver to 10-15% of control 20 (ealine) levels- Fxatnple 13 Screening of ant-isensve oligonutcleouldes a complementary to humkn PXC-71: A series of 20-mer phosphorothioate oligoriucleotides comp~lementary to human PKC-11 were synthesizad. These oligonucleotides were screened at a 35 concentatiofl of S00 n?4 for ability to decrease pKC-1 tq"A levels in human A549 cellx, using a Northern blot aeay.
The oljgonuclaotide sequences are rboi.n in Table 10 and the results are shown in 17igure 11- TABLE oLIaoNucLEOTIItEs TARGRT=D TO [UKAN PKC-11 ui~A ISIS# sequence 0**@OS
S
S
000*
S
S
@0 0 0 6431 CGA CAT GCC 6442 CAG ACG ACA 6443 GCC TGC TTC 6432 ACA GGT GCA 6433 GTC CCG TCT 6435 CCT CAC CGA 15 6441 ATT GAA CTT 6581 TCT CAC TCC 6580 TTC CTT TGG 6e*36 AAC TCQ AGG 6434 CGC CTT CGC 6444 GGA AGO GGT 6445 AAC ACG CCC 6446 GTC TCA AGA 6553 GCG ATG GTT 6605 0CC CTC TCT 6579 CTG GGA ACG 6603 AAG GCT GAT
GGC
TGC
OGA
GOA
CAGC
CAT
CCA
GIrT
ATA
G16T
ATT
TG
CJIG
Caqc
TCC
C7(,n
GCC
CGG
GIC
GTC
GCC
GGA
GGT
TAA
CTC
CCG
GCC
TGC
GCC
CGT
CTG
ACT
GAT
GGG
OCT
CGC
GOA
GAG
AGC
CCC
GCC
GGC
GTG
CCG
CTT
GrIG
CAC
GCT
GC
CCC
AGA
AAG
GC
CG
GA
GC
CICS'
TC
AG
TA
CC
TC
TO
Cc
CA
CG
CC
CA
GG
GT
'arget
AUG
AUG
5. LMTR 5' IUrR
UTR
Coding Coding 3' UTR 3' TJTR Coding Coding Coding Coding Coding Coding 3' t3R 3' UTR 3' UTR SEQ ID
NO:
41 42 43 S4 46 47 48 54 56 57 58 59 61 62 0l1.gonucleotider 6432, 6443, 6431, 6442, 6435, 6434.
644. 6553, 6581 and 6603 reduced PKC-77 mP A levels by greater than 50%. The mosr potent oligonucJeatides were ISIS 6581 (targering 3' untranslared region) and ISIS 6445 (targeting coding region) which gave nearly complete loss of PKC mMNA in thisi assay.
36 Example 14 ScreemIng of a=-iwe a~ oligonuU-lectides cowemetary co iuau flC- t: A series of 2a--mer phoophorotioate oligonilcleor ides complemenrary to htumalri PXC!-f were synthesized as described s in rixample The source of the target "equence was rnbankc locus HSPKCZ, accession numiber Z151138 (Rug, These oligonucleoti.des were screened at a concentrationl of 500 nM for ability to decrease PICC-f zMR~A l.evels in hiuman A549 cells, substantially a5 described in FExample 6 using a Northern blot assay- The oligonucleotide sequences and results of zhe Pcre8Zi are shown in Table 11.
Table 11 IRHISITION OF uRKA EXPRESSION IN UPLMT AS49 CRLLS US INI AWTISESSE
OT
4 IGONUCILEOTIDES COMPLJWWNARY To PxC- Z Oligo 9007 9008 9009 9010 9011 9012 9013 9014 9015 9016 901.7 901B 9019 9U20 9021 9022 9023 9024 9025 3S 9026 3027 9028 9029 #Sequen~ce Target region CGCCGCTCCCTTI'CCACTTG
AUG
CCCCGTAATGCGCCTTGAUG Codlflc CTGTCCACCCACITAGGGT Codiuc OCTTCCTCCATCI'TCTGGCT CodzinE CGGTACAGCTTCCITCCATCT Codiric TTGGAAGAGGTGGCCO~rGG Codinc CCTGTTAAAGCG(4'ITGGCTT Coclinc TGCAGGTCAGCGCGACGAGG Cadini G;CT CTTGCOGAAGCCATGACA Cod izn TTCTTCAACCGCACCAGGAG Codint TTCTTCAACCGC1LCCAGG-AG Codint CTCTGCCTCTGCikTGTGGAA Codial TCCTTGCACATGi!!CGTAGTC Codin', TCCACGCTCAACC-CGTACTC Codini GGAcjCGCCCGGCCATCATCT Codin GGGCTCGCTGGTaAACTGTG Codin' GACGCACOCGGCCTCACACC Stop GGTCAATC-ACGCGTGCC2!A 3' UT TCGGAGCCGTGCCCAGCCTG 31 CCGGGCCA1GGTG IGAGGGACT 3' UT CCGCGACGCAGGCACAGCAG 3' UT TG'GAAACCCCATGACAGCCC 3' tjT GGTCAGTr.CATC:GAGTTCTG
M~
!kInhiib- 70 r 68 1 19 r 3 8 '71 x 41.
59 0 '73 6 3 31.
80 83.
82 70 R 82 40 38 'R 54 'R 79 Seq. ID 63 66 67 69 71 72 73 74 176 77 178 .79 s0 81 82 83 84 37 In this experivent. o].igonuJ4Sotidev .9007, 9008. 9011, 9012, 9013, 9015, 90107, 90181 9020. 9021. 9022, 9021. 9024.
9025, 9028 and 9029 shlowed at least Sot inhibiti.on of m.UNA levels and are presently preferred.
Example 15 Scree=n g of antisenhs oligozrcleotides coupleuieftary to huwm PKC-i£: A 2eries of 20-mer phosphorothicate aligonucl.eotides, compl.ementary to human PKC-E! were syntheie~zed as df-cribed in Example 1. The sour-ce of the targert sequence was Geribank locus RSPI(CE, accession number X65293 (Burns eL These oligonuclecides were screened at a concentration of 500 nM4 for abil1ity to decrease PICC-e tnRNA levelp i~n human A549 cells, substantially as described in Example 6 usiug a N4orthern blot assay- The oligonucleotide seq~uences and 15 resulto of the ocreen are show~n in Table 12- Table 12 IN!SIBITION OF %Ml~i EXPR~ESSION INT HUNAZ A549 CBI43 USINZG ANTISM)SE OLIGONUCLEOTIJDPS COIEPIJ WARY TO PJCC-E MRSA O1go Sequence Target regiov %Inhbib Seq. IP 7933 ACTACCATGGTCCGGGCG= AUG 0 86 7334 GTCCCACCGCATCGGCGC!AGC Coding 0 87 7935 GT'TWCCGATGCGGt3AGTC Coding 0 as *7936 TGCAOTTGGCCAC'GAAGTCG Coding 0 8032 GTGC=GC.ATGTT(.*ACGCTGA Coding 0 8031 CCAGA6GCAGGACCC-ACAGT coding 0 91 '7939 TCTCCTCGGTTGTrCAAATGA Coding 0 92 7940 CGGTGCTCCTc-Ti7CTCGGTT Coding 0 93 7941 AGCCAAAAT1CCT--CTCTG Coding 0 94 7942 CATGAGGGCCGkrGTGACCT Coding 62 7943 ATCCCTTCCTTGCAC-ATCCC Coding 4 96 7944 CCCCAGGOCCCACCAGTCCA Coding 42 97 '79.%5 AICACCCCCAGGCCACCA Coding 56 98 '7949 CGTACATCAGCACCCCCAGG Coding 55 99 7547 CCAGCCATCATCTCGTACAT Coding is 100 7948 TGCCACACAGCC'CAGGCGCA Coding 55 101 7 94 9 TCAGGGCATCACGTCTTCAC Stop a 102 1950 CTCTCArGGCA'IcAGGTCTr Stop 0 103 38 In this excperimenlt, oligonucleotides 7942, 7944, 7945, 7946 and 7948 showed at least 40V inhibition of mR4NA leve3.m and are presently preferred.
Example 16 MU *uequenzg of rue 3' umtralated region of hUM=f PK.Ca A549 Celle (obtained fromu the American Type Culture collection, Bethesda 'MD) were grown to confluence in 6-well plates (Falcon iLabware, Lincoln Park. Na) in Dulbecco's modified Eagie's medium (DME1 containing 1 g glucose/liter and io'k fecal calf serum (F'CS, Irvine Scientific, Santa An&, CA). Celle were harvested and rot~1 RNA was isolated using standard methods. Sambrook, Fritsch, and T_ Maniatis (1989). Mole~cular Cloning: a laboratory manual.
Cold Spring~ Harbor Laboratory, cold spring Harbor, N.Y., Ch~. 7).
cDN'A was made from the RNA using the 3' RACE technique of Frohman et al- LWrohznan. Du~h. M-K. and C.R. Martin (1988) Piroc. liarl. Acad. Sci. U-SA. 85.8.998- .9002) and the 3' RACE~ kit from Gikbco/BRL (Bathesda, MD~) Por making the first strand of cDNA, an oligo dT primer was used. For subsequent amplification from the site of the polyfA) tail, the ol:Lgonuc3.eocide provided in the kit or an Idenrtical oligonucleatide (ISIS 5586; SEQ TD NO: 107; GGCCACGCGTCGrCAGTACrT~TTT=TTTTTTTTTT Fox, amplification from thae interior of the known sequence, TSIS 6288 was used (SEO 11) NO- 108: GGGGTAA!TGCGGCGGCAGTrATGAAACTCACCAGCG The DNA resulting from the PCR reaction was gel-purified, digested with Sal I and Bcl 1, and then cloned into the Bluescript plasmid (Stratagele. La Jolla, CA) using standard techniques (Samnbrook et al-, 1989). The clonad DNJA waa sequenced using a Sequenase Kit from USB.
The new sequence obtained, from the Bcl I site near the 3- end of the previously known sequence (GeriBaxik 3S accesa4.of number x52479) to the most frequently obtained site of polyadenylation is shown as nucleocides 56-1136 1i 39 Figure 12. This site is believed to be the 31 end of th~e -short (4kcb) PKC* iuess2Lge To extamnd this sequence and hence obtain oequencem Apecific. for the long MKa message (8-5 Wcb. zbe technique of Inverse PCR ijas pe:xf rmed. Ocbman, HI., Garber, A.S. and D.L. Hartl. (1988) Genieti.ci 120:1521-623. This5 technique was performed three times using a three sets of primers and rerstri.ctlof enzymes. Each round resulted in about 200 bases of new sequence; the total of the new sequence
(SEQ
ID NO: 104) is rshown in bold type (nucleotidea 1137-1812) in Figure 12. This sequence is showJn exctending in the 3' direction beginning at the Ecl I site (TGATCA) near the end of the previously pub~lished PKCa cDNA sequence.
Finkenzeller et .Nucl. ACi4E Res. 18l:21.83 (1990)'; Genbank accession nunber X52479. Newly determined sequences :7 egin. at nucleotide S6 and are undjlj~ed (SEQ TD NO:105) The most common site of polyadenylation, believed to be the 3' end of the 5hort 1A kcb) mRNA rranscri.pr, i.9 at nucleotide 1136. Sequiences downstream from this site, and therefore unique to the long messatie are in bgi (SPQ ID ND:J.06) Example 17 AnT;Lsense oligonucleoitides targeted to novel sequlencer. in the 3' UTR of PI[Ce A series of phaosphozottiioate antisense oligonucleocides, compl~ementary to the novel sequence obtained as deocribed i.n Example I-G, were designed and synthesp.zed. These oligonucleotides were screened on the basis of their ability to cause the reduction or elimination of PICao RNA in A549 cells 24 hours after the start of treatment. A549 cells were treated with phosphorothioate o).igonucleitides at 500 nM for four hours in the presence of the cationic lipids DOTMA/DOPE, washed and allowed to recover for an additional 20 hours. Total RNA was extracted anid 20kg of each was resolved on 1-2;; gels and transferredi to nylon membranes- These blots were probed with a 32P racdiolabeled PXC-a cDNA probe and then 40 stripped s=4 reprobed with a radiolabeled 03PDH probe to confirmf equal RNA loading. The two major PKC-* transcripts kcb and 4-0 kb) were examined and quantif ied with it PhoaphorImhager (Molecular Dynamics, Sunnyvale The Qligonucleor-ide and zheir activities are shown in Table 13 Table 33 Inhibit ion of PE(Ca mRNA (bothk long and aborrt) by phosphozothioar-e aririsen e oligoiiicleotides (500 nM) IQ Expressed as percent of conitrol mRXX level *aa.
a a a a
ISIBO
7416 '7417 7418 7419 7420 1902 7907 7908 7,909 7911 7912 7913 Sequence Activity MUFgec region CAGTGCCCATOTGCACGrGAG AGAACCTGC1AC.AAATAPGAGC
AGAAACAAGAACCTOC'ACAA
GCAWGATTCAGC~TM1MC
AGGGAGGGAAAGCACILGAAG-
A=GAGGGAAACCACJ
GAAG
TCAGCTCAAAAATAGI'CCTC
CGAAAGGTGACATGAhGAAA GGCGGGAACCA3GACGAA.
GCATGCCACGTlGTGTACC!A TGCAAAACGTATTAAIaTCC TTATAAACATGCAAAAkTTC!A 100# 100% 100% 1001 901r 8 51 2.00%s 901 100;'; PKCt long mRSA PyKCcf long MMNA FKCa long MRNJA PXCoz long mIRNA PKCCf long MA PT(Cc" long mRNA PXCa long ITINA PKC& long mRSA PKCcU long mRNA PxKa long TIRNA PKCa short mRXA PKca 1hort mRNZ.
SEQ
ID NO: 10-9 11.0
III
112 113 113 114 115 116 117 11B 119 25 ISIS 7911 (SEQ ID NO; 117) reduced PKCiX MNiA levelS (both long and short messages) in this preliminary experiment by S0% comrpared to control. Thi.s oliganucleotide i.s therefor~e preferrzed. Further analysis demonstrated that ISIS 7912. selectively reduced the 4mount of long (8.5 kb) message during the first six hours of treatment, with a fourfold oelectivity at 3 hours post-tr~armelt. By 12 hours after treatment w.ith ISIS 7911, level.s of both messages were reduced by over BOV. Timie-course data are sahown in Figure 13~- 41 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Nicholas Dean, C. Frank Bennett and Russell
T.
Boggs (ii) TITLE OF INVENTION: Oligonucleotide Modulation of Protein Kinase C (iii) NUMBER OF SEQUENCES: 119 (iv) CORRESPONDENCE
ADDRESS:
ADDRESSEE: Woodcock Washburn Kurtz Mackiewicz Norris STREET: One Liberty Place 46th Floor CITY: Philadelphia STATE:
PA
COUNTRY:
USA
ZIP: 19103 COMPUTER READABLE FORM: MEDIUM TYPE: DISKETTE, 3.5 INCH, 1.44 Mb STORAGE COMPUTER: IBM PS/2 OPERATING SYSTEM:
PC-DOS
SOFTWARE: WORDPERFECT 5.1 (vi) CURRENT APPLICATION
DATA:
APPLICATION NUMBER: n/a FILING DATE: herewith
CLASSIFICATION:
(vii PRIOR APPLICATION
DATA:
APPLICATION NUMBER: 852,852 FILING DATE: March 16, 1992 APPLICATION NUMBER: 08/089,996 FILING DATE: July 9, 1993 42 APPLICATION NUMBER: 08/199,779 FILING DATE: February 22, 1994 (viii) ATTORNEY/AGENT
INFORMATION:
NAME: Rebecca Ralph Gaumond REGISTRATION NUMBER: 35,152 REFERENCE/DOCKET NUMBER: ISIS-1546 (ix) TELECOMMUNICATION
INFORMATION:
TELEPHONE: (215) 568-3100 TELEFAX: (215) 568-3439 INFORMATION FOR SEQ ID NO: 1: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CCCCAACCAC CTCTTGCTCC INFORMATION FOR SEQ ID NO: 2: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GTTCTCGCTG GTGAGTTTCA INFORMATION FOR SEQ ID NO: 3: 43 SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AAAACGTCAG CCATGGTCCC INFORMATION FOR SEQ ID NO: 4: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GGATTCACTT CCACTGCGGG INFORMATION FOR SEQ ID NO: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) -SEQUENCE DESCRIPTION: SEQ ID NO: GAGACCCTGA ACAGTTGATC INFORMATION FOR SEQ ID NO: 6: SEQUENCE
CHARACTERISTICS:
44 LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CCCGGGAAAA CGTCAGCCAT INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: o TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CTGCCTCAGC GCCCCTTTGC INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: *AGTCGGTGCA GTGGCTGGAG INFORMATION FOR SEQ ID NO: 9: SEQUENCE
CHARACTERISTICS:
LENGTH: 45 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GCAGAGGCTG GGGACATTGA INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid *o STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GGGCTGGGGA GGTGTTTGTT INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CACTGCGGGG AGGGCTGGGG INFORMATION FOR SEQ ID NO: 12: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid 46 STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: AGCCGTGGCC TTAAAATTTT INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: ATTTTCAGGC CTCCATATGG INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: AAGAGAGAGA CCCTGAACAG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid 47 STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GATAATGTTC TTGGTTGTAA INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single 33t3* t" TOPOLOGY: linear.
(iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: ATGGGGTGCA CAAACTGGGG INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 9 (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GTCAGCCATG GTCCCCCCCC INFORMATION FOR SEQ ID NO: 18: 'SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single 48 TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CGCCGTGGAG TCGTTGCCCG INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TCAAATGGAG GCTGCCCGGC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear e (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TGGAATCAGA CACAAGCCGT INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 49 (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: CATCTTGCGC GCGGGGAGCC INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION; SEQ ID NO: 22: TGCGCGCGGG GAGCCGGAGC INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CGAGAGGTGC CGGCCCCGGG INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: S(B) TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes 50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: CTCTCCTCGC CCTCCGTCGG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TGGAGTTTGC ATTCACCTAC INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: AAAGGCCTCT AAGACAAGCT INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: 51 GCCAGCATGT GCACCGTGAA INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: ACACCCCAGG CTCAACGATG INFORMATION FOR SEQ ID NO:,29: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: CCGAAGCTTA CTCACAATTT INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ACTTAGCTCT TGACTTCGGG 52 INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: ATGCTGCGGA AAATAAATTG INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: ATTTTATTTT GAGCATGTTC INFORMATION FOR SEQ ID NO: 33: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: TTTGGGGATG AGGGTGAGCA INFORMATION FOR SEQ ID NO: 34: 53 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: CCCATTCCCA CAGGCCTGAG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGGAGCGCGC CAGGCAGGGA INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: CCTTTTCCCA GACCAGCCAT INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: 54 LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: GGCCCCAGAA ACGTAGCAGG INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: GGATCCTGCC TTTCTTGGGG INFORMATION FOR SEQ ID NO: 39: *e SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: 'CAGCCATGGC CCCAGAAACG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 55 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGACATGCCG GCGCCGCTGC INFORMATION FOR SEQ ID NO: 41: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
S**
CAGACGACAT GCCGGCGCCG INFORMATION FOR SEQ ID NO: 42: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: GCCTGCTTCG CAGCGGGAGA INFORMATION FOR SEQ ID NO: 43: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid 56 STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: ACAGGTGCAG GAGTCGAGGC INFORMATION FOR SEQ ID NO: 44: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: GTCCCGTCTC AGGCCAGCCC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCTCACCGAT GCGGACCCTC INFORMATION FOR SEQ ID NO: 46: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single 57 TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: ATTGAACTTC ATGGTGCCAG INFORMATION FOR SEQ ID NO: 47: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes o. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: *o TCTCACTCCC CATAAGGCTA INFORMATION FOR SEQ ID NO: 48: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: TTCCTTTGGG TTCTCGTGCC INFORMATION FOR SEQ ID NO: 49: SEQUENCE CnARACTERISTICS: LENGTH: TYPE: nucleic acid ST-ANDEDNESS: single TOPOLOGY: linear v' ANTI-SENSE: yes (x4' SEQUENCE DESCRIPTION: SEQ ID NO: 49: TTCCATCCTT CGACAGAGTT INFORMATION FOR SEQ ID NO: Wi SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear ANTI-SENSE. e SEQUEN _CE DESCRIPTION. SEQ ID NO: *AGGCTGA'"TGC TGGGAAGGTC I-NFORYNLkT7ON FOR SEQ ID NO: 51: ()SEQUENCE_
CHARACTERISTICS:
LENGTH: ()TYPE: nucleic acid TOPOLOGY: linear v) AN\TI-SENSE: yes SEQUENCE DESCRIPTION. SEQ D NO: 51: GTTCT _GGC TGATGCTGGIG INO2L.~NFOR SEQ ID NO: 52: SEQUENCE CHLAR-kCTERISTICS: ENT:19 TYPE: nucleic acid SC 7R X.'DEDNESS: single TOPOLOGY: linear (iANTI-S7:SE VeS 59 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: TTCTCGCTGG TGAGTTTC 18 INFORMATION FOR SEQ ID NO: 53: SEQUENCE
CHARACTERISTICS:
LENGTH: 17 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: TCTCGCTGGT GAGTTTC 17 INFORMATION FOR SEQ ID NO: 54: *o SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: AACTCGAGGT GGCCGCCGTC INFORMATION FOR SEQ ID NO: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60
S
4*
S
S
S.
5 *5S*
S
SSS
S
5.5.
a CGCCTTCGCA TAGCCCTTTG INFORMATION FOR SEQ ID NO: SE: Wi SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS. single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: GGAAGGGGTJG ATTGCGGGCC INFORXIATION FOR SEQ ID NO: 57: SEQUENCE CHAkRACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) AINTI-SENSE: yes (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: AACACGCCCA TTGCCCACCA INFOR.KkTION FOR SEQ ID NO: 58: SEQUENCE
CHARACTERISTICS:
LENGTh':: TYPE: nucleic acid ST.3-1 DEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE.: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: GGCGTGCTCG 61
S
5 o* A S S INFORMATION FOR SEQ ID NO: 59: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION:
SE
GCGATGGTTC
AGCTGGGCCC
INFORMATION FOR SEQ ID NO: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION:
SEQ
GCCCTCTCTC TCACTCCCCA 2 INFORMATION FOR SEQ ID NO: 61: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION:
SEQ
CTGGGAAGGT CCGATAGAGG INFORMATION FOR SEQ ID NO: 62: Q ID NO: 59: ID NO: 0 ID NO: 61: 62 SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: AAGGCTGATG CTGGGAAGGT INFORMATION FOR SEQ ID NO: 63 SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: CGCCGCTCCC TTCCATCTTG INFORMATION FOR SEQ ID NO:64 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: CCCCGT.AATG CGCCTTGAGG INFORMATION FOR SEQ ID NO: SEQUENCE
CHARACTERISTICS:
-63 (A)i LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE. DESCRIPTION: SEQ ID NO: CTGTCCACCC ACTTGAGGGT INFORMATION FOR SEQ ID NO: 66: Wi SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic agid STRANDEDNESS: single 0 TOPOLOGY: linear (iv) AmNTI-SE7NSE: yes (xi4) SEQUENCE DESCRIPTION: SEQ ID NO: 66: GCTTCC-TCC.A TCTTCTGGCT :No>:o7FOR SEQ ID NO: 67: OSOS(iJ) SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic a i STRAINDEDNESS. single TOPOLOGY: linear ANTI-SENSE: yes (xi4) SE--QUENITCE DESCRIPT7"ION: SEQ ID NO: 67: CZ=1,AA=T TCCTCCATCT FOR SEQ ID NO: 68: SE=QUE7NCE= CHAkRACTERISTICS: LENGTH:
S
S
S
S
64 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (x4) SEQUENCE DESCRIPTION: SEQ ID NO: 68 TTGGAAGAGG TGGCCGTTGG INFORL{kTION FOR SEQ ID NO: 69: Wi SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS. single TOPOLOGY: linear (iV) ALN::-s7-_E: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: CCTGTTA.AAG CGCTTGGCTT iNFORITAT:7ON FOPR SEQ ID NO: SEQUENCE CH4ARACTERISTICS: LENGTH: TYPE: nucleic acid STRA.NDEDNESS: single TOPOLOGY: linear (iv) ANT:--SENSE yes Wx) SEQUENCE DESCRIPTION: SEQ ID NO: TGCAGGTCAG CGGGACGAGG INFOR M-TTO1N FOR SEQ ID NO: 71: SQUE-NCE CtHaRAC7E-7STIC-S: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: GCTCTTGGGA AGGCATGACA INFORMATION FOR SEQ ID NO: 72: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: TTCTTCAACC GCACCAGGAG INFORMATION FOR SEQ ID NO: 73: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: TTCTTCAACC GCACCAGGAG INFORMATION FOR SEQ ID NO: 74: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single 66 (iv) (xi) TOPOLOGY: lirleaz ANTI-SENSE: yes SEQUENCE
DESCRIPTION
CTCTGCCTCT
GCATGTGGA
C
INFORMATION FOR SEQ ID NO: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STPANDEDNESS: single TOPOLOGY: linear,, (iv) ANTI-SENSE: yes (XiL) SEQUENCE DESCRIPTION:
SE'
TCCTTGCACA
TGCCGTAGTC
!INFORMATION FOR SEQ ID NO: 76: SEQUENCE C~kAPACTERISTICS: LEN3=: TYPE: nucleic acid STRATNDEDNESS. single TOPOLOGY: linear (iv) ANTI-SENSE. yes (xi) SEQUENCE DESCRIPTION:
SEQ
TCCACGCTGA ACCCGTACTC 2 I2NFORMALTION FOR SEQ ID NO: 77: Ni) 'SEQUENCE Cl~kpACTERISTICS: LENTG::: TYPE": nucleic acid STRANDEDNESS: single EQ ID NO: 74: Q ID NO: ID NO: 76: 67
C.
C
C
TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: GGAGCGCCCG GCCATCATCT INFORMATION FOR SEQ ID NO: 78: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: GGGCTCGCTG GTGAACTGTG INFORMATION FOR SEQ ID NO: 79: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: GACGCACGCG GCCTCACACC INFORMATION FOR SEQ ID NO: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear *5*S
S
*SS.
*5 S S
S.
*SS.
S
S
S..
S
S
(iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GGGTCAATCA CGCGTGTCCA INFORMATION FOR SEQ ID NO: 81: Ci) SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes SEQUENCE DESCRIPTION: SEQ ID NO: 81: TCGGAGCCGT GCCCAGCCTG INFORM/ATION FOR SEQ ID NO: 82: SE1QUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid C) TANDEDN-SS: sinale TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: CGGGCCAGGT GTGAGGGACT IFO-3yI7 FOR SEQ ID NO: 83: SEQUENCE CHARAzCTERISTICS: LEINGTH: -()TYPE: nutcleic acid STRAN*DEDNESS: singin D)TOPOLOGY: linear (iv) N::-SENSE: yes -69 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83: CCGCGACGCA GGCACAGCAG INFORMATION FOR SEQ ID NO: 84: Wi SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: TGGAAACCGC ATGACAGqCC INFORMA.TIONT FOR SEQ ID NO: UI) SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid S. S:RANDEDNESS: single .PLCy.'nar (iv) _NTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: SGGTCAGTGCA TCGAGTTCTG INFORMY11,ION FOR SEQ ID NO: 86: SEQUENCE CHARAzCTERISTICS: LENGTH: YPE:nucleic acid SITDEDNESS: single TOPOLOGY: linear (i)ANTI-SENSE: yes (xi)1 SEQUENCE DESCRIPTION: SEQ ID NO: 86: 70 555...
S
*5
S
*5 S S
S
S
ACTACCATGG TCGGGGCGGG INFORMATION FOR SEQ ID NO: 87: Wi SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: E GTCCCACCGC ATGGCGCAGC INFORMATION FOR SEQ ID NO: 88: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) AN7I-SENSE: yes (X4) SEQUENCE DESCRIPTION: SEQ ID NO: 88 GTTTGGCCGA TGCGCGAGTC iNFORNIATION FOR SEQ ID NO: 89: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: Plucleic acid STRADEDNESS: single TO'POLOGY: linear (iv)A T~SENE: es (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: TGCAGTTGGC CACQ-A)GTCG 7 71 INFORMATION FOR SEQ ID NO: Wi SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNEgg: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTGGGGCATG TTGACGCTGA INFORMATION FOR SEQ ID NO: 91: SEQUENCE
C~{ARACTERISTICS:
LENGTH: TYPE: nucleic acid STRkNDEDNESS: single TOPOLOGY: linear (iv) ANT I -SEN yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: *.CCAGA -_CAGG GACCCACAGT INFORMATION FOR SEQ ID NO: 92: too-: SEQUENCE
CHARACTERISTICS:
LENGTH-: TYPE: nucleic acid (CI) STRANDEDNESS: single TOPOLOGY: linear (L'qATTISE'NSEZ: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92: TCTCCTCGGT TGTCAAATGA INFORMAzTION FOP. SEQ ID NO: 93:
S
*5*S
S
5555 *5 S S *5 S S *5 S *5
S
*S*S
S
S
S
72 SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS. single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGGTGCTCCT CTCCTCGGTT INFORMVATION FOR SEQ ID NO: 94: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94 AGC~AATCCTCTTCTCTG INFOR,%:TION FOR SEQ ID NO: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STPAIJDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE. yes (xiy, SEQUENCE DESCRIPTION: SEQ ID NO: CATGAGGGCC GATGTGACCT !NFORMA:TION FOR SEQ ID NO: 96: SEQUENCE
CH.AR.ACTERISTICS:
93 73
O.W
00.0 .00 000.
C.
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTP-SE-NSE: yes (xi) SEQUENCE DESCRIPTION:
SE
ATCCCTTCC
TTGCACATCCC
INFORMVATION FOR SEQ ID NO: 97: Ci) SEQUE-NCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION:
SEQ
CCCCAGGil-GCC CACCAGTCCA 2 INFORj~MATION FOR SEQ ID NO: 98: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TO0POLOGY: linear Ci v) ANTI-SENSE: yes CXi) SEQUENZE DESCRIPTION:
SEQ
ACCACCCA GGGCCCACCA 2 !N7FR2VLT7-N FOR SEQ 7D NO: 99: SEQUENCE
CHARACTERISTICS:
CA) LENGTH: ~Q ID NO: 96: ID NO: 97: 1D NO: 98: 74 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99: CGTACATCAG CACCCCCAGG, INFORMATION FOR SEQ ID NO: 100: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iJv) ANTI-SENSE: yes (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100: CCAGCCATCA TCTCGTACAT INFOR1M,1T7ON FOR SEQ ID NO: 101: (iJ) SEQUNC
CH{ARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE. yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101: TOCCACACAIG CCC.AGGCGC.A INFOVL--TION FO-R SEQ ID NO: 102: SE-QUENCE7
CG-LZLACTERISTICS.
LENGTH: TYPE7: vnucleic- acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102: TCAGGGCATC AGGTCTTCAC INFORMATION FOR SEQ ID NO: 103: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103: CTCTCAkGGGC ATCAGGTCTT INFOR.KkTION FOR SEQ ID NO:104: SEQUENCE
CLARACTERISTICS:
(A LENGTHE: 18'12 base pairs TYPE: nucleic acid STRMTDEDNESS: double TOPOLOGY: linear (iMOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: TGATCAACTG TTCAGGGTCT CTCTCTTACA ACCAAGAACA TTATCTTAGT
GGAAGATGGT
ACGTCAT'JCT CGGCA TT2ATT.LCTG TAGAAGTTAC GTLCTGG-1CTCT
AGGTTAACCC
120 TTCCTLAG.k-AA GCAAGCAGAC TGTTGCCCCA TTTTGGGTAC AATTTGATAT
ACTTTCCATA
180 -76 CCCTCCATT GTGGATTTT CAGCATTGGA ATCCCCCAC CAGAGATGTT
AAAGTGAG;CT
240 GTCCCAGA ACATCTCCc CCAAGACGTC TTTGGAATCC AAGAACAGGA
AGCCAAGAGA
300 GTGAGCAGG AGGGATTGGG GGTGGGGGGA GGCCTCAAA TACCGACTGC
GTCCATTCTC
360 TGCCTCCATG GAAACAGCCC CTAGAATCTG AAAGGCCGGG ATAAACCTA
TCACTGTTCC
420 CAAACATTGA CAAATCCTA CCCAACCATG GTCCAGCAGT TACCAGTTTA
AACAAAAA
480 ACCTCAGATG AGTGTTGGGT GAATCTGTCAL TCTGGTACCC TCCTTGGTTG
ATAACTGTCT
TGTCTT AAGAGGCCAA ATCGTCTAAG GACGTTGCTG
AACAAGCGTG
600 TGATCT TCAGATCAAG GATAAGCCAG TGTGTACATA TGTTCATTTT
AATCTCTGGG
660 AGATTATTTT TCCATCCAGG GTGCCATCAG TAAkTCATGCC ACTACTCACC
AGTCG:TGTTC
720 GCCAACACCC ACCCCCACAC ACACCAACAT TTTGCTGCCT ACCTTGTTAT
CCTTCTCAAG
S 780 AAGCTGAAGT GTACGCCCTC TCCCCTTTTG TGCTTATTTA TTTAATAGGC
TGCAGTGTCG
840 CTTATGAG TACGATGTAC AGTAACTTAA TGGAAGTGCT GACTCTAGCA
TCAGCCTCTA
900 CCGATTGAT f TTCCTCCCTT CTCTAGCCCT GGATGTCCAC TTAGGGATAA
AA-AGAATATG
960 GTTTTGGTTC CCATTTCTAG TTCACGTTGA ATGACAGGCC TCGAGCTGTA
GAATC.AGG-A
1020 77 44t* 4 .4 4* 4 4 4449 .444 ACCCGGATGC CTAACAGCTC AAAGATGTTT TGTTAATAGA AGGATTTTAA
TACGTTTITGC
1080 AAATGCATCA TGCAATGAAT TTTGCATGTT TATAATAAAC CTTAATAACA
AGTGAATAGA
1140 AGGATTTTAA TACGTTTTGC AAATGCATCA TGCAATGAT TTTGCATGTT
TATAATAC
1200 CTTAATAACA AGTGAATCTA TATTATTGAT ATAATCGTAT CAAGTATA
GAGAGTATTA
1260 TAATAATTTT ATAAGACAA ATTGTGCTCT ATTTGTGCAG GTTCTTGTTT
CTAATCCTCT
1320 TTTCTAATTA AGTTTTAGCT GAATCCCTTG CTTCTGTGCT TTCCCTCCCT
GCACATGGGC
1380 ACTGTATCAG ATAGATTACT TTTTAAATGT AGATAAAATT TCAAAAATGA
ATGGCTAGTT
1440 TACGTGATAG ATTAGGCTCT TACTACATAT GTGTGTGTAT ATATATGTAT
TTGATTCTAC
1500 CTGCAACAA ATTTTTATTG GTGAGGACTA TTTTTGAGCT GACACTCCCT
CTTAGTT:-CT
1560 TCATGTCACC TTTCGTCCTG GTTCCTCCGC CACTCTTCCT CTTGGGGACA
ACAGGAAGTG
1620 TCTGATTCCA GTCTGGCCTA GTACGTTGGT ACACACGTGG CATTGCGCAG
CACCTGGGICT
1680 GACCTTTGTG TGTAGCGTGT GTGTGTGTTT CCTTCTTCCC TTCAGCCTGT
GACTC-TTGCOT
1740 GACTCCAGGG GTGGGAGGGA TGGGGAGACT CCCCTCTTGC TGTGTGTACT
GGACACGCAG
1800 GAAGCATGCT
GA
1812 -78 INFORMATION FOR SEQ ID NO:105: Wi SEQUENCE
CHARACTERISTICS:
LENGTH: 1757 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULJE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: ATGGTACGTC ATGCTCAGTG TCCAGTTTAA TTCTGTAGAA GTTACGTCTG
GCTCTAGGTT
AACCCTTCCT AGAAAGCAAG CAGACTGTTG CCCCATTTTG GGTACAATTT
GATATACTTT
120 CCATACCCTC CATCTGTGGA TTTTTCAGCA TTGGAATCCC CCAACCAGAG
ATGTTAAAGT
180 .*GAGCTGTCCC AGGAAACATC TCCACCCAAG ACGTCTTTGG AATCCAAGAA
CAGGAAGCCA
240 AGAC-AGTGAG CAGGGAGGGA TTGGGGGTGG GGGGAGGCCT CAAAA:-ACCG
ACTGCG"TCCA
300 TTCTCTGCCT CCATGGAAAC AGCCCCTAGA ATCTGAAAGG CCGGGATA.
CCTAATCACT
360 GTTCCCAAAC ATTGACAAAT CCTAACCCAA CCATGGTCCA GCAGTTACCA GTTTAAAOCz~ 420 AAAAAACCTC AGATGAGTGT TGGGTGAATC TGTCATCTGG TACCCTCCTT GGTTC-ATAkC 480 TGTCTTGATA CTTTTCATTC TTTGTAAGAG GCCAAAkTCGT CTAAGGACGT TGCTG-AkCAA 540 GCGTGTGAAA TCATTTCAGA TCAAGGATAA GCCAGTGTGT ACATATGTTC
ATTTTAATCT
600 79 a.* *6 CTGGGAGATT ATTTTTCCAT CCAGGGTGCC ATCAGTAATC ATGCCACTAC
TCACCAGTGT
660 TGTTCGCCAA CACCCACCCC CACACACACC AACATTTTGC TGCCTACCTT
GTTATCCTTC
720 TCAAGAAGCT GAAGTGTACG CCCTCTCCCC TTTTGTGCTT ATTTATTTAA
TAGGCTGCAG
780 TGTCGCTTAT GAAAGTACGA TGTACAGTAA CTTAATGGAA GTGCTGACTC
TAGCATCAGC
840 CTCTACCGAT TGATTTTCCT CCCTTCTCTA GCCCTGGATG TCCACTTAGG
GATAAAAAGA
900 ATATGGTTTT GGTTCCCATT TCTAGTTCP C GTTGAATGAC AGGCCTGGAG
CTGTAGAATC
960 AGGAAACCCG GATGCCTAAC ACCTCAAAGA TGTTTTGTTA ATAGAAGGAT
TTTAATACGT
1020 TTTGCAAATG CATCATGCAA TGAATTTTGC ATGTTTATAA. TAAACCTTAA
TAACAAGTGA
1080 ATAGA.AGGAT TTTAATACGT TTTGCAAATG CATCATGCAA. TGAATTTTGC
ATTLCTTATAA
1140 TAAACCTTAA TAACAAGTGA ATCTATATTA TTGATATPAT CGTATCAAGT
ATAAAGAGAG
1200 TATTATAATA ATTTTATAAG ACACAATTGT GCTCTATTTG TGCAGGTTCT
TGTTTCTAAT
1260 CCTCTTTTCT AATTAAGTTT TAGCTGAATC CCTTGCTTCT GTGCTTTCCC
TCCCTGCACA
1320 TGGGCACT T ATCAGATAGA TTACTTTTTA AATGTAGATA AAkATTTCAAA
AATC-AATGGC
1380 TAGTTTACGT GATAGATTAG GCTCTTACTA CATATGTGTG TGTATATATA
TGTATTTGAT
1440 TCTACCTGA AACAAATTTT TATTGGTGAG GACTATTTTT GAGCTGACAC
TCCCTCTTAG
1500 TTTCTTCATG TCACCTTTCG TCCTGGTTCC TCCGCr-CTC TTCCTCTTGG
GGACAACAG
1560 AAGTGTCTGA TTCCAGTCTG GCCTAGTACG TTGGTAAC CGTGGCATTG
CGCAGCACCT
1620 GGGCTGACCT TTGTGTGTAG CGTGTGTGTG TGTTTCCTTC TTCCCTTCAG
CCTGTGACTG
1680 TTGCTGACTC CAGGGGTGGG AGGGATGGGG AGACTCCCCT CTTGCTGTGT
GTACTGGAA
1740 CGCAGGAAGC
TCG
1757 INFORMATION FOR SEQ ID NO:106: SEQUENCE
CHARACTERISTICS:
to LENGTH: 676 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: TAGAAGGATT TTAATACGTT TTGCAAATGC ATCATGCAAT GAATTTTGCA
TGTTTATAAT
AAACCTTAT AACAAGTGAA TCTATATTAT TGATATAATC GTATCAAGTA
TAAAGAGAGT
120 ATTATAATAA TTTTA1TAAGA CACAATTGTG CTCTATTTGT GCAGGTTCTT
GTTTCTAATC
-180 CTCTTTTCTA ATTAAGTTTT AGCTGAATCC CTTGCTTCTG TGCTTTCCCT
CCCTGCACAT
240 GGGCACTGTA TCAGATAGAT TACTTTTTAA ATGTAGATA AATTTCAAA
ATGAATGGCT
300 AGTTTACGTG ATAGATTAGG CTCTTACTAC ATATGTGTGT GTATATATAT
GTATTTGATT
360 CTACCTGCAA ACAAATTTTT ATTGGTGAGG ACTATTTTTG AGCTGACACT
CCCTCTTAGT
420 TTCTTCATGT CACCTTTCGT CCTGGTTCCT CCGCCACTCT TCCTCTTGGG
GACAACAGGA
480 AGTGTCTGAT TCCAGTCTGG CCTAGTACqT TGGTACACAC GTGGCATTGC
GCAGCACCTG-
540 S GGCTGACCTT TGTGTGTAGC GTGTGTGTGT GTTTCCTTCT TCCCTTCAGC
CTGTGACTGT
600 TGCTGACTCC AGGGGTGGGA GGGATGGGGA GACTCCCCTC TTGCTGTGTG
TACTGGACAC
660 GCAGGAAGCA
TCG
676 INFORMVATION FOR SEQ ID NO: 107: Wi SEQUENCE
CHARACTERISTICS:
LENGTH: 37 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: no~ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: GGCCACGCGT CGACTAGTAC TTTTTTTTTT TTTTTTT 37
I
82 INFORMATION FOR SEQ ID NO: 108: SEQUENCE
CHARACTERISTICS:
LENGTH: 37 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: no (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108: GGGGTAGAAT GCGGCGGCAG TATGAAACTC ACCAGCG 37 INFORMATION FOR SEQ ID NO: 109: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109: CAGTGCCCAT GTGCAGGGAG INFORMATION FOR SEQ ID NO: 110: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110: AGAACCTGCA CAAATAGAGC INFORMATION FOR SEQ ID NO: 111: 83 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111: AGAAACAAGA ACCTGCACAA INFORMATION FOR SEQ ID NO: 112: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112: C GCAAGGGATT CAGCTAAAAC INFORMATION FOR SEQ ID NO: 113: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi- SEQUENCE DESCRIPTION: SEQ ID NO: 113: AGGGAGGGAA AGCACAGAAG INFORMATION FOR SEQ ID NO: 114: SEQUENCE CHARACTERISTICS: 84 LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114: TCAGCTCAAA AATAGTCCTC INFORMATION FOR SEQ ID NO: 115: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115: CGAAAGGTGA CATGAAGAAA INFORMATION FOR SEQ ID NO: 116: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: GGCGGAGGAA CCAGGACGAA INFORMATION FOR SEQ ID NO: 117: SEQUENCE
CHARACTERISTICS:
LENGTH: 85 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117: GCAATGCCAC GTGTGTACCA INFORMATION FOR SEQ ID NO: 118: SEQUENCE
CHARACTERISTICS:
LENGTH: TYPE: nucleic acid fe*E* a STRANDEDNESS: siqgle TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118: TGCAAAACGT ATTAAAATCC INFORMATION FOR SEQ ID NO: 119: SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119: TTATAAACAT GCAAAATTCA 86 THE CLAIMS DEFINING THE INVENTON ARE AS FOLLOWS 1. An oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-e gene or mRNA, wherein said oligonucleotide includes a nucleotide sequence selected from the group consisting of SEQ ID NO: 95, 97, 98, 99 and 101.
2. The oligonucleotide of claim 1 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate.
3. The oligonucleotide of claim 1 wherein at least one of the nucleotides comprises a modification on the 2' position of the sugar.
15 4. The oligonucleotide of claim 1 which is a chimeric oligonucleotide.
15 5. A pharmaceutical composition comprising the oligonucleotide of claim 1 and a pharmaceutically acceptable carrier or diluent.
6. An oligonucleotide having up to 50 nucleotide units specifically 20 hybridizable with a protein kinase C-a gene or mRNA. wherein said S" oligonucleotide comprises a nucleotide sequence selected from the group Sconsisting of SEQ ID NO:52 and 53.
7. The oligonucleotide of claim 6 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate.
*e 8. The oligonucleotide of claim 6 wherein at least one of the nucleotides comprises a modification on the 2' position of the sugar.
9. The oligonucleotide of claim 6 which is a chimeric oligonucleotide.
A pharmaceutical composition comprising the oligonucleotide of claim 6 and a pharmaceutically acceptable carrier or diluent.
11. An oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-r7 gene or mRNA, wherein said

Claims (52)

12. The oligonucleotide of claim 11 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate.
13. The oligonucleotide of claim 11 wherein at least one of the nucleotides comprises a modification on the 2' position of the sugar.
14. The oligonucleotide of claim 11 which is a chimeric oligonucleotide. A pharmaceutical composition comprising the oligonucleotide of claim 11 and a pharmaceutically acceptable carrier or diluent.
16. An oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-S gene or mRNA. wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:63. 64. 67. 68. 69, 71. 73. 74. 76. 77. 78. 79, 80. 81. 84. and 20 85.
17. The oligonucleotide of claim 16 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate.
18. The oligonucleotide of claim 16 wherein at least one of the nucleotides comprises a modification on the 2' position of the sugar.
19. The oligonucleotide of claim 16 which is a chimeric oligonucleotide. A pharmaceutical composition comprising the oligonucleotide of claim 18 and a pharmaceutically acceptable carrier or diluent.
21. A method of modulating the expression of protein kinase C in cells comprising contacting the cells with an oligonucleotide having up to about nucleotide units. said oligonucleotide being specifically hybridizable with a 88 protein kinase C-e gene or mRNA, wherein said oligonucleotide includes a nucleotide sequence selected from the group consisting of SEQ ID NO: 95, 97, 98, 99 and 101.
22. The method of claim 21 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate.
23. The method of claim 21 wherein at least one of the nucleotides of said oligonucleotide comprises a modification on the 2' position of the sugar.
24. The method of claim 21 wherein said oligonucleotide is a chimeric oligonucleotide.
25. A method of modulating the expression of protein kinase C in cells comprising contacting the cells with an oligonucleotide having up to about nucleotide units. said oligonucleotide being specifically hybridizable with a protein kinase C-c gene or mRNA. wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:52 and 53. 20
26. The method of claim 25 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate.
27. The method of claim 25 wherein at least one of the nucleotides of said oligonucleotide comprises a modification on the 2' position of the sugar. .i
28. The method of claim 25 wherein said oligonucleotide is a chimeric oligonucleotide.
29. A method of modulating the expression of protein kinase C in cells comprising contacting the cells with an oligonucleotide having up to about nucleotide units, said oligonucleotide being specifically hybridizable with a protein kinase C-qr gene or mRNA. wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:54. 55. 56.
57. 58. 59. 60, 61. and 62. 89 The method of claim 29 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate. 31. The method of claim 29 wherein at least one of the nucleotides of said oligonucleotide comprises a modification on the 2' position of the sugar. 32. The method of claim 29 wherein said oligonucleotide is a chimeric oligonucleotide. 33. A method of modulating the expression of protein kinase C in cells comprising contacting the cells with an oligonucleotide having up to about nucleotide units, said oligonucleotide being specifically hybridizable with a protein kinase C-5gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:63, 64. 67, 1 5 68. 69. 71. 73. 74. 76. 77. 78. 79. 80. 81, 84. and 34. The method of claim 33 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate. 20 35. The method of claim 33 wherein at least one of the nucleotides of said oligonucleotide comprises a modification on the 2' position of the sugar. 36. The method of claim 33 wherein said oligonucleotide is a chimeric oligonucleotide. 37. A method of detecting in a sample the presence of a protein kinase C-a gene or mRNA comprising contacting the sample with an oligonucleotide having up to 50 nucleotide units specifically hybridizable with said gene or mRNA, wherein said oligonucleotide includes a nucleotide sequence selected from the group consisting of SEQ ID NO: 95, 97, 98. 99 and 101 and detecting hybridization. 38. A method of detecting in a sample the presence of a protein kinase C-a gene or mRNA comprising contacting the sample with an oligonucleotide having up to 50 nucleotide units specifically hybridizable with said gene or mRNA. wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:52 and 53. and detecting hybridization. 39. A method of detecting in a sample the presence of a protein kinase C-7 gene or mRNA comprising contacting the sample with an oligonucleotide having up to 50 nucleotide units specifically hybridizable with said gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:54, 55, 56, 57, 58, 59, 60, 61, and 62, and detecting hybridization. A method of detecting in a sample the presence of a protein kinase C- gene or mRNA comprising contacting the sample with an oligonucleotide having up to 50 nucleotide units specifically hybridizable with said gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 63. 64. 67, 68. 69. 71. 73, 74. 76. 77, 78. 79. 80, 81.
84. and 85, and detecting hybridization. 41. The method of claim 40 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate. 42. The method of claim 40 wherein at least one of the nucleotides of 2 said oligonucleotide comprises a modification on the 2' position of the sugar. 43. A method of treating a condition associated with expression of protein kinase C comprising administering to a mammal or cells thereof with a therapeutically effective amount of an oligonucleotide having up to 50 nucleotide 25 units specifically hybridizable with a protein kinase C-e gene or mRNA. wherein said oligonucleotide includes a nucleotide sequence selected from the group consisting of SEQ ID NO: 95, 97. 98, 99 and 101. 44. The method of claim 43 wherein said condition is a hyperproliferative disorder. The method of claim 43 wherein said hyperproliferative disorder is psoriasis. 46. The method of claim 43 wherein said hyperproliferative disorder is colorectal cancer. 47. The method of claim 43 wherein said hyperproliferative disorder is lung cancer. 48. The method of claim 43 wherein said hyperproliferative disorder is breast cancer. 49. The method of claim 43 wherein said hyperproliferative disorder is skin cancer. The method of claim 43 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate. 51. The method of claim 43 wherein at least one of the nucleotides of said oligonucleotide comprises a modification on the 2' position of the sugar. 52. The method of claim 43 wherein said oligonucleotide is a chimeric oligonucleotide. 2 53. The method of claim 43 wherein said oligonucleotide is in a pharmaceutically acceptable carrier or diluent. a. S a a 54. cationic lipid. The method of claim 53 wherein said carrier or diluent comprises a A method of treating a condition associated with expression of protein kinase C comprising administering to a mammal or cells thereof with a therapeutically effective amount of an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C-a gene or mRNA. wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:52 and 53. 56. The method of claim 55 wherein said condition is a hyperproliferative disorder. 57. The method of claim 56 wherein said hyperproliferative disorder is psoriasis. 58. The method of claim 56 wherein said hyperproliferative disorder is colorectal cancer. 59. The method of claim 56 wherein said hyperproliferative disorder is lung cancer. 60. The method of claim 56 wherein said hyperproliferative disorder is breast cancer. 61. The method of claim 56 wherein said hyperproliferative disorder is skin cancer. 62. The method of claim 55 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate. 63. The method of claim 55 wherein at least one of the nucleotides of 2C said oligonucleotide comprises a modification on the 2' position of the sugar. 64. The method of claim 55 wherein said oligonucleotide is a chimeric oligonucleotide. 25 65. The method of claim 55 wherein said oligonucleotide is in a pharmaceutically acceptable carrier or diluent. C C 66. cationic lipid. The method of claim 65 wherein said carrier or diluent comprises a 67. A method of treating a condition associated with expression of protein kinase C comprising administering to a mammal or cells thereof with a therapeutically effective amount of an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C- q gene or mRNA. wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:54. 55. 56. 57. 58. 59. 60. 61. and 62. 93 68. The method of claim 67 wherein said condition is a hyperproliferative disorder. 69. The method of claim 68 wherein said hyperproliferative disorder is psoriasis. The method of claim 68 wherein said hyperproliferative disorder is colorectal cancer. S 71. lung cancer. 72. breast cancer. 73. skin cancer. The method of claim 68 wherein said hyperproliferative disorder is The method of claim 68 wherein said hyperproliferative disorder is The method of claim 68 wherein said hyperproliferative disorder is 2 74. The method of claim 67 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate. The method of claim 67 wherein at least one of the nucleotides of said oligonucleotide comprises a modification on the 2' position of the sugar. 76. The method of claim 67 wherein said oligonucleotide is a chimeric oligonucleotide. 77. The method of claim 67 wherein said oligonucleotide is in a pharmaceutically acceptable carrier or diluent. 78. cationic lipid. The method of claim 77 wherein said carrier or diluent comprises a 79. A method of treating a condition associated with expression of protein kinase C comprising administering to a mammal or cells thereof with a therapeutically effective amount of an oligonucleotide having up to 50 nucleotide units specifically hybridizable with a protein kinase C- gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:63, 64, 67, 68, 69, 71, 73, 74, 76, 77, 78, 79, 80, 81, 84, and
85. The method of claim 79 wherein said condition is a hyperproliferative disorder. 0 81. The method of claim 80 wherein said hyperproliferative disorder is psoriasis. 82. The method of claim 80 wherein said hyperproliferative disorder is colorectal cancer. a 83. lung cancer. The method of claim 80 wherein said hyperproliferative disorder is 84. The method of claim 80 wherein said hyperproliferative disorder is breast cancer. The method of claim 80 wherein said hyperproliferative disorder is skin cancer.
86. The method of claim 79 wherein at least one of the intersugar linkages between nucleotide units of the oligonucleotide is a phosphorothioate.
87. The method of claim 79 wherein at least one of the nucleotides of said oligonucleotide comprises a modification on the 2' position of the sugar.
88. The method of claim 79 wherein said oligonucleotide is a chimeric oligohucleotide.
89. The method of claim 79 wherein said oligonucleotide is in a pharmaceutically acceptable carrier or diluent. The method of claim 89 wherein said carrier or diluent comprises a cationic lipid.
91. A method of diagnosing a condition associated with protein kinase C comprising contacting a sample from a mammal suspected of having a condition associated with protein kinase C with an oligonucleotide having up to nucleotide units specifically hybridizable with a protein kinase C-e gene or mRNA, wherein said oligonucleotide includes a nucleotide sequence selected from the group consisting of SEQ ID NO: 95, 97, 98, 99 and 101 and detecting hybridization.
92. The method of claim 91 wherein said condition is a hyperproliferative disorder.
93. The method of claim 92 wherein said hyperproliferative disorder is 15 psoriasis, colorectal cancer, lung cancer. breast cancer, or skin cancer. 94 A method of diagnosing a condition associated with protein kinase C comprising contacting a sample from a mammal suspected of having a condition associated with protein kinase C with an oligonucleotide having up to 20 nucleotide units specifically hybridizable with a protein kinase C-a gene or mRNA. wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 52 and 53. and detecting hybridization. The method of claim 94 wherein said condition is a 25 hyperproliferative disorder.
96. The method of claim 95 wherein said hyperproliferative disorder is psoriasis, colorectal cancer, lung cancer, breast cancer, or skin cancer.
97. A method of diagnosing a condition associated with protein kinase C comprising contacting a sample from a mammal suspected of having a condition associated with protein kinase C with an oligonucleotide having up to nucleotide units specifically hybridizable with a protein kinase C-qr gene or mRNA. wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:54, 55, 56. 57, 58, 59. 60. 61, and 62. and detecting hybridization. 96
98. The method of claim 97 wherein said condition is a hyperproliferative disorder.
99. The method of claim 98 wherein said hyperproliferative disorder is psoriasis, colorectal cancer, lung cancer, breast cancer, or skin cancer.
100. A method of diagnosing a condition associated with protein kinase C comprising contacting a sample from a mammal suspected of having a condition associated with protein kinase C with an oligonucleotide having up to nucleotide units specifically hybridizable with a protein kinase C-4 gene or mRNA, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:63, 64, 67, 68, 69, 71, 73, 74, 76, 77, 78, 79, 80, 81. 84. and 85, and detecting hybridization. S15
101. The method of claim 100 wherein said condition is a hyperproliferative disorder.
102. The method of claim 101 wherein said hyperproliferative disorder is 20 psoriasis. colorectal cancer, lung cancer, breast cancer, or skin cancer.
103. An isolated nucleic acid molecule comprising a sequence substantially homologous to the sequence set forth in SEQ ID NO:105. 25 104. The nucleic acid molecule of claim 103 wherein said nucleic acid molecule is comprised of deoxyribonucleic acid subunits.
105. The nucleic acid molecule of claim 104 wherein said nucleic acid molecule is double-stranded.
106. An isolated nucleic acid molecule comprising a sequence substantially complementary to the sequence set forth in SEQ ID NO:105.
107. An antisense oligonucleotide up to 50 nucleotides in length comprising a nucleotide sequence specifically hybridizable with a portion of the sequence set forth in SEQ ID NO:105. A4 97
108. A pharmaceutical composition comprising the oligonucleotide of claim 107 and a pharmaceutically acceptable carrier.
109. A polynucleotide probe comprising a nucleotide sequence specifically hybridizable with a portion of the nucleic acid molecule of claim 103.
110. A polynucleotide probe comprising a nucleotide sequence specifically hybridizable with a portion of the nucleic acid molecule of claim 106.
111. An antisense oligonucleotide up to 50 nucleotides in length comprising a nucleotide sequence which is specifically hybridizable with the long m RNA transcript of human protein kinase C-a and which is not specifically hybridizable with the short mRNA transcript of human protein kinase C-a.
112. The antisense oligonucleotide of claim 111 comprising a nucleotide sequence specifically hybridizable with a portion of the sequence set forth in SEQ ID NO:106. 20 113. The oligonucleotide of claim 112 comprising the sequence set forth in SEQ ID NO:117.
114. A pharmaceutical composition comprising an oligonucleotide of claim 112 and a pharmaceutically acceptable carrier.
115. A polynucleotide probe comprising a nucleotide sequence specifically hybridizable to the long mRNA transcript of human protein kinase C-a.
116. The polynucleotide probe of claim 115 comprising a nucleotide sequence specifically hybridizable with a portion of the sequence set forth in SEQ ID NO: 106.
117. The polynucleotide probe of claim 126 comprising a sequence as set forth in SEQ ID NO:117. -,J 98
118. A method for detecting a gene coding for human protein kinase C-a in a sample comprising contacting the sample with a polynucleotide probe of claim 117 or claim 118 under conditions which allow for the formation of a polynucleotide duplex between the probe and said gene coding for protein kinase C-a; and detecting the presence or absence of a polynucleotide duplex whereby the presence of a polynucleotide duplex indicates the presence of said gene coding for human protein kinase C-a in said sample.
119. A method for detecting the long mRNA transcript of human protein kinase C-a in a sample comprising contacting the sample with the polynucleotide probe of claim 123 under conditions which allow the formation of a polynucleotide duplex between the probe and the long mRNA transcript of human protein kinase C-c and detecting the presence or absence of a polynucleotide duplex whereby the presence of a polynucleotide duplex indicates the presence of said long mRNA 15 transcript of human protein kinase C-a in said sample. f
120. A method for modulating the expression of protein kinase C-a in a cell containing a protein kinase C-a gene comprising contacting the cell with an antisense oligonucleotide up to 50 nucleotides in length, said antisense 20 oligonucleotide comprising a nucleotide sequence specifically hybridizable with a portion of the sequence set forth in SEQ ID NO:105. se.
121. A method for specifically modulating the expression of the long mRNA transcript of protein kinase C-a in a cell containing a protein kinase C-c gene comprising contacting the cell with an antisense oligonucleotide up to nucleotides in length, said antisense oligonucleotide comprising a nucleotide sequence specifically hybridizable with a portion of the sequence set forth in SEQ ID NO:106.
122. A method of treating an animal having a condition associated with protein kinase C-a comprising contacting said animal with a therapeutically effec'tive amount of an antisense oligonucleotide up to 50 nucleotides in length. said antisense oligonucleotide comprising a nucleotide sequence specifically hybridizable with a portion of the sequence set forth in SEQ ID NO:105. 99
123. A method of treating an animal having a condition associated with expression of protein kinase C-a comprising contacting said animal with a therapeutically effective amount of an antisense oligonucleotide up to nucleotides in length, said antisense oligonucleotide comprising a nucleotide specifically hybridizable with a portion of the sequence set forth in SEQ ID NO: 106. see**: *0 0 Abstract Compositions and methods are provided for the treatment and diagnosis of diseases associated with protein kinase C. Oligonucleotides are provided which are specifically hybridizable with a PKC gene or mRNA. Oligonucleotides specifically hybridizable with a particular PKC isozyme, set of isozymes or mRNA transcript are provided. Methods of treating conditions amenable to therapeutic intervention by modulating protein kinase C expression with an oligonucleotide specifically hybridizable with a PKC gene or mRNA are disclosed. Compositions and methods are provided for the treatment, detection and diagnosis of diseases associated with protein kinase C alpha and specific transcripts thereof. New nucleic acid sequences are provided which encode 3' untranslated regions of human protein kinase C alpha. Polynucleotide probes for PKC alpha are also disclosed. *000 Goes 0000 0 *0 0 o•
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US08/089,996 US5703054A (en) 1992-03-16 1993-07-09 Oligonucleotide modulation of protein kinase C
US089996 1993-07-09
US08/199,779 US5681747A (en) 1992-03-16 1994-02-22 Nucleic acid sequences encoding protein kinase C and antisense inhibition of expression thereof
US199779 1994-02-22
AU73981/94A AU688354B2 (en) 1993-07-09 1994-07-08 Oligonucleotide modulation of protein kinase C

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANTISENSE RES AND DEVELOPMENT(1991) VOL 1,P 35-42 *
EXPERIMENTAL CELL RESEARCH(1993) VOL 205, P 52-58 *
THE JOUR OF BIOL CHEM (1993) VOL 268, P 11946-11950 *

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