CA2488792A1 - Antisense oligonucleotides for treatment of cancer - Google Patents
Antisense oligonucleotides for treatment of cancer Download PDFInfo
- Publication number
- CA2488792A1 CA2488792A1 CA002488792A CA2488792A CA2488792A1 CA 2488792 A1 CA2488792 A1 CA 2488792A1 CA 002488792 A CA002488792 A CA 002488792A CA 2488792 A CA2488792 A CA 2488792A CA 2488792 A1 CA2488792 A1 CA 2488792A1
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- Prior art keywords
- carcinoma
- antisense
- sarcoma
- antisense oligonucleotide
- use according
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
Abstract
RI.alpha. antisense oligonucleotides and pharmaceutical compositions thereof are disclosed. Methods for treating certain cancers in animals comprising administering to animals an effective amount of an RI.alpha. antisense oligonucleotide, or a pharmaceutical composition thereof, are also disclosed.
Description
-la-ANTISENSE OLIGONUCLEOTIDES FOR TREATMENT OF CANCER
FIELD OF THE INVENTION
The invention is in the field of medicinal chemistry. In particular, the invention relates to certain antisense oligonucleotides and the use thereof for the treatment of cancer.
BACKGROUND OF THE INTENTION
Control mechanisms for cell growth and differentiation are disrupted in neoplastic calls (Potter, V.R. (1988) Adv.~Oncol. 4, 1-8; Strife, A. &
Clarkson, B. (1988) Semin. Hematol. 25, 1-19; Sachs, L. (1987) Cancer Res. 47, 1981-1986). cAMP, an~
intracellular regulatory agent, has been considered to have a role in the control of cell proliferation and differentiation (Pastan, I., Johnson, G.S. & Anderson, W.B. (1975) nn. Rev. Biochem. 44, 491-522; Prasad, K.N. (1975) Biol. Rev. 50, 129-165; ~Cho-Chung, X.S.
(1980) J. Cyclic Nucleotide Res. 6, 163°-177; Puck, T.T. (1987) Somatic Cell Mot. Genet._ 13, 451-457).
Either inhibitory or stimulatory effects of cAIKP on cell growth have been reported previously in studies in which cAMP analogs such as N6-OZ ~-dibutyryladenosine 3',5~-cyclic monophosphate or agents that raise ... 2 _ intracellular cAMP to abno.rznal and continuously high levels were used, and available data are interpreted.
very 'differently (chapowski, F.J., Kelly, L.A. &
Butcher, R.W. (1975) Adv. Cyclic Nucleotide Protein Phosphorylat. Res: 6, 245-338; Cho-Chung, Y.S. (1979) in Influence of Hormones on Tumor Development, eds.
Kellen, J.A. & Hilf, R. (CRC, Boca Raton, FL), pp. 55-93); Prasad, K.N. (1981) in The Transformed Cell, eds.
Cameron, L.L. & Pooh, T.B. (Academic, New York), pp.
235-266; Boynton, A.L. & Whit~ield, J.F. (1983) Aav.
Cyclic Nucleotide Res 15, 193-294).
Recently, site-selective CAMP analogs were discovered which show a preference for binding. to purified preparations of type II rather than type-' I
cAMP-dependent protein kinase in vitro (Robinson-Steiner, A.M. & Corbin, J.D. (1983) J. Biol. Chem.
258, 1032-1040; f6greid, D., Ekanger, R., Suva, R.K., Miller, J.P., Sturm, P., corbin, J. D. & Deskeland, S.O. (1985) Eur. J. Biochem. 150, 2i9-227), provoke potent growth inhibition, differentiation,'and reverse transformation in a broad spectrum of human and rodent cancer cell lines (Katsaros, D., Tortora, G., Tagliaferri, P., Clair, T., Ally, S., Neckers, L., Robins, R.K. & Cho-Chung, Y.S. (1987) FEBS Lett. 223, 97=103; Tortora, G_, Tagliaferrz, P., Clair, T., Colamonici, O., Neckers, L_M., Robins, R.K. & Cho-Chung, Y..S. (1988) B oo , 71, 230-233; Tagliaferri "
F., Katsaros, D., Clair, T., Robins,. R.K. & Cho-Chung, Y.S. (1988) J. Biol. Chem. 263, 409-416). The type I
and type II protein kinases are distinguished by their regulatory subunits (RI and RII, respectively) (Corbin, J.D., Keely, S.L. & Park, C.R. (1975) 3.
Biol. C~em. 250,- 218-225; Ho~mann, F., Beavo, J.A. &
Krebs, E.G. (1975) J. Biol. Chew. 250, 7795-7801).
3-5 Four different regulatory subunits.[RI« (previously designated RI) (Lee, D.C., Carmichael, D.F., Krebs, E.G. & McXnight, G.S. (1983) Proc. Natl. Acad. Sci.
USA 8a, 3608-3612y, RTE (Clegg, C.i., Cadd, G.G.
&
McKnight, G.S. (i988j Proc. Natl. Aced. Sci. USA
85, 3703-3707), RTxa (RTT~y) (Scott, J.D., Glaccum, M.B., Zoller, M.J., Uhler, M.D., Hofmann, D.M., McXnight, G.S. & Krebs, E.G. (1987) Proc Natl Agad Sci LISA
84, 5192-5195) and ,RII~ (RII51) (Jahnsen, T., Iiedin, h., Kidd, V.J., Beattie, W.G., Lohmann, S.M., Walter, U., Durica, J., Schul2, T.Z., Schlitz, E., Browner, M., Irawrence, C,B:; Goldman, D., Ratoosh, S.k.
&
Richards, J.S. {1986) J. Biol. Chem. 261, 12352-12361)] have now been identified at the gene/mRNA
' level. Two different catalytic subunits [Ca (Lmler, M.D., Carmichael, D.F., Lee, D.C. Chrivia, J.C., Krebs, F.G. & McKnight, G.S. {x986) Proc. Natl.
Aced.
Sci. USA 83, 1300-1304) and C~ (Uhler, M.D,., Chrivia, .
261, J,C. ~& McKnight, G.S. (1986) ~T. Biol~, Chem.
15360-15363; Showers, M,o. & Maurer, R.A. (1986) J.
Siol. Chem. 261, 16288-16291)3 have also been identified; however, preferential coexpression of ~ either one of these catalytic subunits with either the type I or type II protein kinase regulatory subunit has not been found (Showers, M.o. & Maurer, R.A.
(1986) J. Biol. Chew: ~6x, 16288-16291).
The growth inhibition by site-selective cAl~
anatlogs parallels reduction in RIa with an increase in RII~, resulting in an increase of the RII~/Rx ratio in cancer cells (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros,, D., f~greid, D., D6skelarid, S.O., Jahnsen, T, & Cho-Chung, Y.S.
(1988) ~oc_ Natl. Aced. Sci. USA $~, 6319-6322; Gho-Chung, Y.S. (1989) J. Natl. Cancer Inst. 81, 982-987).
Such selection modulation of RIa versus RII~ is riot mimicked by ~:reatment with N6,02~-dibutyryladeno-sine 3~,5~-cyclic monophosphate, a,previously studied a 38 CAMP analog (Ally, S., Tortara, G.,~
Clair, 2~., Grieco, D. , Merlo, G. , ICatsaros, D. , Qlgreid, D. , Deskel:and, S.O., Jahnsen, T. & Cho-Chung, Y.S. (1.988) .Prop.
Nat7.w, ... 4 _ Aced. Sci. USA 85, 6319-6322). The growth inhibitian further correlates with a rapid translocation of RII~
to the nucleus and an increase in the transcription of the RII~ gene (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros, D., ggreid, D., Dgskeland, S.O., Jahnsen, T. & Cho-Chung, Y.S.
(1988) proc. Natl. Aced. Sci. USA 85,-6319-6322). These results support the hypothesis that RII~ plays an important role in the cAMP growth regulatory function {Cho--Chung, Y.S. (1989j J. Natl. Cancer Inst.
81, 982-987).
Antisense RNA sequences have been described as naturally occurring biological inhibitors of gene expression in both prokaryates (Mizuno, T., Chou, M-Y, arid Inouye, M. {1984), Proc. Natl. Aced. Sci.
USA 81, (1966-1970)) and eukaryotes (Heywood, S.M. Nucleic Acids Res., 14, 6771-67?2 (2986)), and these sequences presumably function by hybridizing to complementaz-y mRNA sequences, resulting in hybridization arrest of translation (Peterson, B.M., Roberts, B.E., and ICuff, E.L., {1977) Proc. Natl. Aced. Sci. USA, ?4, 4374. Antisense oligodeoxynucleotides are short synthetic nucleotide seguences formulated to be complementary to a specific gene or RNA message.
.
Through the binding of these oligomers to a target DNA
or mRNA sequence, transcription or translation of the gene carr be selectively blocked and the disease process generated by that gene can be halted.
The cytoplasmic location of mRNA provides a target 38 considered to be readily accessible to antisense oligodeoxynucleotides entering the cell; hence much of the work in the field has focused on RNA as a target.
Currently, the use of antisense oligodeoxynucleotides provides a useful tool for exploring regulation of .35 gene expression in vitro and in tissue culture (Rothenberg, M., Johnson, G.; Laughlin, C., Green, I., Craddock, d., Sarver, N., and Cohere, 3'.S.(1989j J.
Natl. Cancer Inst., 83:1539--1544.
SUhQ2ARY OF TFiE INVEPi'PION
The invention is related to the discovery that inhibiting the expression of RIa in leukemia cells by contact with an antisense o-oligonucleotides and S-oligonucleotides fo~'~RI results in the inhibition of proliferation and the stimulation of cell differentiation. Accordingly, the invention is directed to RIa antisense oligonucleotides and phar~aaceutical compositions thereof for the treatment of cancer.
In particular, the invention is related to 15-to 3o-mer antisense oligonucleotides which ~. are complementary to a region in the first 1,00 N-terminal codons of RIa (Seq. ID No:6j.
The invention is also related to 15- to 3o-mer antisense oligonucleotides which are a fragment of antisense DNA complementary to RI (Seq. ID No:
5j.
The invention is also related to pharmaceutical compositions comprising at least one 15- to 30-mer antisense oligonucleotide which is complementary to a region in the first 10o N-terminal codons of RIa (Seq.
ID No:6); and a pharmaceutically acceptable carrier.
The~invention is also related to a method for treating cancer by suppressing growth of cancer cells susceptible to growth suppression and for inducing cancer cell differentiation in an animal comprising administering to an animal in need of such treatment 3o a cancer cell growth suppressing amount of an RIa antisense oligonucleotide.
DESCRIPTIO~1 of ,~E F3,C~,URES
Fiq. 1 depicts a graph showing the effect of RId antisense oligodeoxynuclentide on the basal rata of growth of HL-60 leukemia cells {A} and the growth of these cells when treated with cAM.P analogs or TPA (B}.
A, cells were,grown {sae the Examples} in the absence (o} or presence (~} of RIa antisense oligodeoxy-nucleotide (15 ACM}. At indicated'~times, cell counts in duplicate were performed. Data represent the average values -r SD of four experiments: I3, on day ~
of experiment A, cells exposed or unexposed to RIB
antisense oligodeoxynucleotide were reseeded (day 0}
at 5 x los cellsJdish, and cells pre-exposed to RIQ
antisense oligodeoxynucleotide were further treated with the oligomer at day 0 and day 2. cAMp analogs and. TPA were added one time at day o. Cell counts were performed on a Coulter counter on day 4. 8-Cl, 8~C1-cAMP (10 ;.cM}; 8-C1 + N6-B, 8-C1-cAMP {5 ACM} + N6-benzyl-cAMP (5 ~M}; TPA (3.0 ~ M}_ The data represent the average values ~ SD of four experiments.
Fia. 2 depicts a graph showing the effect of RIa antisense oligodeoxynucleotide on the morphologic transformation of HL-60 cells. Cells either exposed or unexposed to RIa antisense oligodeoxynucleotide were treated with cAMP analogs or TPA as described in Fig. 1B. On day 4 (see Fig. 1B}, cells were washed twice in Dulbecco's phosphate-buffered saline and were pelleted onto a glass slide by cytocentrifuge. The resulting cytopreparations were fixed and stained by Wright's stain. x Z80.
Fia~s depicts a Northern blot showing decreased RIQ
mRNA expression in HL-60 leukemia cells exposed to RIa antisense oligodeoxynucleotide. Cells were either exposed or unexposed to RI« antisense oligadeoxynucleotide (15 ACM} for 8 hr. Isolation of total RNA and Northern plot analysis followed the methods described in the Examples. A, ethidium bromide staining of RNA; M, markers of ribosomal RNAs;
lanes l, 2, cells une~tposed or exposed to RIa antisense oligomer. .8, Northern blot analysis; the same nitrocellulose filter eaas hybridized to both RIa and actin probes in sequential manner: .Lanes Z, 2, cells unexposed or exposed to RIB antisense oligomer.
Fiq: 4 depicts an SDS-PAGE showing the~effect of RIa antisense oligodeoxynucleotide on the basal and induced levels of RI and RII~ cAMP receptor proteins in HL-60 leukemic cells. Cells were either exposed to RIa antisense oligodeoxynucleotide (15 ~CMj or treated with cAMP analogs as described in Fig. 1. Preparation of cell extracts, the photoactivated incorporation of g-N3-~32P]CAMP and immunoprecipitation using the anti-RIa or anti-RII~ antiserum and protein A Sepharose, and SDS-PAGE of solubilized antigen-antibody complex followed the methods described in the Examples.
Pre-immune serum controls were carried out simultaneously and detected no immunoprecipitated band. M, labeled marker proteins of known molecular weight;
RIQ, the 48,000 molecular weight RI (Sigma);
RIIa, the 56,000 molecular weight RII (Sigma). Lanes RIa and RII~ are from photoaffinity labeling with 8-N3-[32P]CAMP only; lanes 1 to 3, photoaffinity labeling with 8-N3-[s2P]CAMP followed by immunoprecipitation with anti-RI or anti-RII~ antiserum.. 8-C1, 8-Cl-cAMP
(5 ;CMj; N6-benzyl,Nfi-benzyl-eAMP (5~CMj. The data in the table represent quantification by densitometric scanning of the autoradiograms. The data are expressed relative to the levels in control cells unexposed to RI~~antisense oligomer and untreated with cAMP analog, which are set equal to_1 arbitrary unit_ ' The data represent an average SD of three a5 experiments. A and B, immunoprecipitation with anti-RIa and anti.-RII~ antisera, respectively.
g Ficx. 5 depicts graphs showing the growth inhibition of human cancer cell lines by RIQ antisense oligodeoxynucleotide having SEQ 7D No: 1 (O-oligo and S-oligo derivatives), compared to controls. Cell lines: SK-N-SH, neuroblastoma; T~S-174T, colon carcinoma; MCF-7, breast carcinoma; TMK-l, gastric carcinoma. E2, estradiol-17~.
Fict. s depicts the -change in morphology of 'sK-N-SA
human neuroblastoma sells exposed to RIQ antisense 1.0 oligodeoxynucleotide having SEQ ID No: 1..
Fiq. 7 depicts a graph showing that RIa antisense oligadeoxynucleotide and its phosphorothioate analog inhibit the in vivo growth of LS-174T human. colon carcinoma in athymic mice. Figure 7A shows. the 1.5 oligodeoxynucleotide concentration-dependent inhibition of tumor growth. a-oligo, RI« antisense oligodeoxynucleotide~ S-oligo, phosphorothioate analog of RIa antisense oligomer. The cholesterol pellets (total weight 20 mg) containing the indicated doses of 20 O-oligo or S-oligo were implanted s.c. one time, at zero time, and tumor sizes were measured. Tumor volume (see Materials and Methods, Example 3j represents an average ~ S.D. of 7 tumors. . Figure 7B
shows the temporal effect of antisense 25 oligodeoxynucleotide phosphorothioate analogs on tumor growth. S-oligos as indicated at 0.3 mg dose in cholesterol pellets (total weight 20 mg) were implanted s.c. 2x/week, and tumor volume (see Materials and Methads, Example 3) represents an 30 average 1- S.D. of 7 tumors.
-g-DESCRIPTION OF THE PREFERRED EMBODIMENTS
Antisense therapy is the administration of exogenous oligonucleotides which bind to a target polynucleotide located within the cells. The term "antisense" refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g. RI«. See for example, Jack Cohen, OLIGODEOXYNUCLEOTIDES, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The RIa antisense oligonucleotides of the present invention include derivatives such as S-aligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, s-upra) which exhibit enhanced cancer cell growth inhibitory action (see Figures 5 and 7A).
S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention may be prepared by treatment of the corresponding 0-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide which is a sulfur transfer reagent.
See Iyer, R.P. et al., J. Org. Chem. 55: 4693-4698 (1990); and Iyer, R.P. et al., J. Am. Chem. Soc.
112:1253-1254 (1990).
The RIa antisense oligonucleotides of the present invention may be RNA or DNA which is complementary to and stably hybridizes with the first 100 N-terminal codons of the RIa genome or the corresponding mRNA. Use of an oligonucleotide complementary to this region allows for the selective hybridization to RIa mRNA and not to mRNA specifying other regulatory subunits of protein kinase.
Preferably, the RIa antisense oligonucleotides of the present invention are a 15 to 30-mer fragment of the antisense DNA molecule having SEQ ID N0:5 which hybridizes to RIa mRNA.
Alternatively, RIa antisense oligonucleotide is a 15-to 30-mer oiigonucleotide which is complementary to a region in the first 100 N-terminal codons of RI« (Seq.
ID No:6). Most preferably, the RI« antisense oligonucleotide has SEQ ID No: 1, SEQ ID No: 2, SEQ
ID No: 3, or SEQ ID No: 4.
Included as well in the present invention are pharmaceutical compositions comprising an effective amount of at least one of the RT« antisense oligonucleotides of the invention in combination with a pharmaceutically acceptable carrier. In one embodiment, a single RIa antisense oligonucleotide is utilized. In another embodiment, two RIa antisense oligonucleotides are utilized which are complementary to adjacent regions of the RIa genome. Administration of two RIa antisense oligonucleotides which are complementary to adjacent regions of the RIa genome or v corresponding mRNA may allow for more efficient inhibition of RIa genomic transcription or mRNA
translation, resulting in more effective inhibition of cancer cell growth.
Preferably, the RIa antisense oligonucleotides is coadministered with an agent which enhances the uptake of the antisense molecule by the cells. For example, the RI« antisense oligonucleotide may be combined with a lipophilic cationic compound which may be in the form of liposomes. The use of liposomes to introduce nucleotides into cells is taught, for example, in U.S. Patent Nos. 4,897,355 and 4,394,448. See also U.S. Patent Nos. 4,235,871, 4,231,877, 4,224,179, 4,753,788, 4,673,567, 4,247,411, 4,814,270 for general methods of preparing liposomes comprising biological materials.
Alternatively, the RI« antisense oligonucleotide may be combined with a lipophilic carrier such as any one of a number o~ sterals including cholesterol, cholate and deoxycholic acid. A preferred sterol is cholesterol.
In addition, the RIa antisense oligonucleotide may be conjugated to a peptide that is ingested by cells. Examples of useful peptides include peptide .
and peptide toxins.
hormones, antigens or antibodies, By choosing a peptide that is selectively taken up by the neoplastic cells, specific delivery of the antisense .agent may be effected. The Rl~ antisense oligonucleotide may be covalently bound via the 5'OH.
group by formation of an activated aminoalkyl derivative. The peptide of choice may then be aovalen~ly attached to the activated RIa antisense al'igonucleotide via an amino and sulfhydryl reactive hetero bifunctional reagent. The latter is hound to a cysteine residue present in the peptide_ upon exposure of cells to the RIa antisense oligonucleotide bound to the peptide, the peptidyl. antisense agent is endocytosed and the RIa antisense oligonucleotide binds to the target RTa mRNA to inhibit translation.
See PCT Application Publication No. PCT/US89/02363.
As antineoplastic agents, the RIa antisense 0ligonucleotides of the present invention.are useful .
in txeating a variety of cancers, including, but not limited ta, gastric, pancreatic, lung, breast, anal, colorectal, head and neck neoplasms, neurablastomas, melanoma and various leukemias. ' The RIa antisense oligonucleotides of the invention may also be active against the following tumor systems: F9 teratocarcinoma, SK-N-SH
neuroblastoma, TMIC-1 gastric carcinoma, HL-60 promyelocytic I:eukemia, Leukemia L-3.210, Leukemia p388, P1534 leukemia, Friend Virus:,Leukemia, Leukemia L4946, Mecca lymphosarcoma, Gardner lymphosarcoma, Ridgway Osteogenic sarcoma, sarcoma 180 (ascites), Wagner osteogenic sarcoma, Sarcoma T241, Lewis lung carcinoma, Carcinoma 755, CDSF, MCF-7 breast carcinoma, Colon 38, LS-7.7~1T colon carcinoma, Carcinoma 1025, Ehrlich carcinoma (ascites & solid}, Krubs 2 carcinoma (ascites), Bashford carcinoma 63, Adenacarcinoma E 0771, B16 Melanoma, Hardin-Passey melanoma, Gilama 26, Miyona adenocarcinoma, Walker carcinosarcoma 256, Flexner-Job3ing carcinoma, Jensen sarcoma, Iglesias ~,arcoma, Iglesias ovarian tumor, Murphy-stern lymphosarcoma, Yoshida sarcoma, Dunning leukemia, Rous chicken sarcoma, and Crabb hamster sarcoma.
The RIB antisense oligonucleotides and the pharmaceutical compositions of the present invention may be administered by any means that achieve their intended purpose. For example, administration may be by ' parenteral, subcutaneous, intravenous, intramuscular, intxa-peritoneal, or ~transdermal routes. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
Compositions within the scope of this invention include all compositions wherein the RIa antisense oligonucleotide is contained in an amount which is effective to achieve inhibition of proliferation and/or stimulate differentiation of the subject cancer cells. While-individual needs vary, determination of optimal ranges of effective amounts of each component is with the skill of the art. Typically, the RIa antisense aligonucleotide. may be administered to mammals, e.g. humans, at a dose of 0.005 to 1 mg/kg/day, or an equivalent amount of the pharmaceuti-cally acceptable salt thereof, per day of the body weight of the mammal being treated:.
25 In addition to administering the RIa antisense oligonucleotides as a raw chemical in solution, the RI« antisense oligonucleotides may be administered as part of a pharmaceutical prepaxation containing suitable pharmaceutically acceptable carriers comprising exoipients and auxiliaries. which facilitate processing of the RIB antisense oligonualeotxde into preparations which can be used pharmaceutically.
Suitable formulations y for parenteral administration include ac,~ueous solutions of the Rxa antisense oligonucie~t~.des in water-soluble form.
far example, water-soluble salts. In addition, to . suspensions of the active compounds as appropriate oily . inj ection suspensions ,, may .~ be adnninistexed.
suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oii, or ~ synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
a5 Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxyiaethyl cellulose, soxbitol, and/or dextran. optionally, the suspension may also captain stabilizers.
20 The antisense oligonucleetides of the present invention may be prepared according to any of the methods that are well known to those o ordinary skill.
in the art. Preferably, the antisense ~
phase olir~onuoleotides are prepared by so3:id 25 synthesis. See, Goodchild, J. , ~3ioeon-~uaate Chem's, ~,,:165-167 X1990) , for a review of the chemical synthesis of ~olagonucleotides.
Alternative7~y,~ the antisense oligonualeotides can be obtained from a number of companies which specialize 30 in the custom synthesis of oligonueleotides. .
Having now generally described this invention, c the same will, be vx~darstood by reference to an example which is provided herein for purposes of illustration only and ~is not intending to _be~ limited unless 35 otherwise specified.
- i4 -EXAMPLES
~xamt~le 1 Oligodeoxynucleotides. The 2i-mer oligodeoxynucleo-tides used in the present studies were synthesized at Midland Certified Reagent Co. (Midland, TX) and had the following sequences: human.-RIa .(Sandberg, M., Tasken, K., Oyen, o., Hansson, V. & Jahnsen, T. {1987) Biochem. Biophys. ,f. Res. Common. X49, 939-945) antisense, 5~-GGC-GGT-ACT-GCC--AGA-CTC-CAT-3 ~ (SSQ ID
No:l); human RII~g (Levy, F.O., oyen, O., Sandberg, M., Tasken, K., Eskild, W., Hansson, V. & Jahnsen, T.
(1988) t~1_ Endocrinol., 2, 1364-1373) antisense 5'-CGC-CGG-GAT-CTC-GAT-GCT-CAT-3' (SEQ ID N0:8);
human RIIa Oyen, O., Mykelbust, F., Scott, J.D., Hansson, V. & Jahnsen, T. (1989) FEES Lett. 246, 57-64) antisense 5'-CGG-GAT-CTG-GAT-GTG-GCT-CAT-3' (SEQ ID No:9); and the random sequence oligodeoxynucleotide was made of a mixture of all four nucleotides at every position.
Cell tirowth Experiment. Cells grown in suspension culture in RPM1 1640 medium supplemented with l0% heat-inactivated fetal bovine serum, penicillin (50 U/ml), streptomyc3.n (500 ~ag/atl.), and 1 mM;glutamine (Gibco, Grand Island, NY) were seeded at x 105 cells per dish. Oligodeoxynueleotides were added after seeding and every 48 hr thereafter. Ceil counts were perfonced on a. Coultsr counter. Cells unexposed or exposed to oligodeoxynucleotides for 4 days were reseeded (day O) at 5 x z05 cells/dish, and cells pre-exposed to the oligodeoxynucleotide were further treated with the oligomer at day 0 and day 2.
eAMp analogs (kindly provided by Dr. R.K. Robins, Nucleic Acid Research Institute, Costa Mesa, CA) or 12-O-tetradecanoylghorbol-i3-aceta~,e (TPA3 were added one time at day 0. Cell counts were performed on day 4.
- is -Immunoprecipitation of RIa and RZI~ cA,MP Receptor Proteins after Photoaffinity Labeling with 8-A3-t32p~c~,. Cell extracts were prepared at 0-4°C. The cell pellets (2 x 104 calls) , after two washes with PBS, were suspended in 0.5 ml buffer Ten (0.1 M NaCl, 5 mM MgCl2, 1$ Nonidet P-40, 0.5% Na deoxycholate, 2 KIU/ml bovine aprotinin, and 20 inM Tris-IiCl, pH 7. 4 j containing proteolysis inhibitors (Tortora, G., Clair, T. & Cho-Chung, Y.~~~S. (1990) Proc Natl Acad. Sci.
~ 87, 705-708}, vortex-mixed, passed through a 22-gauge needle l0 times, allawed to stand for 3o min at 4°C, and centrifuged at 750 x g for 20 min; the resulting supernatants were used as,cell lysates. The photoactivated incorporation of 8-N3-[32P]cAMP (60.0 Ci/mmol}, and the immunoprecipitation using the anti-RIa or anti-RII~ antiserwn (kindly provided by Dr. S _ O.
Dgskeland, Universitg of Bergen, Bergen, Norway) and protein A Sepharose~ and SDS-PAGE of solubilized antigen-antibody complex followed the method previously described (Tortora, G., Clair, T. & Cho-Chung, Y. S. (1990) Proc. Natl. Aced. Sci. USA 87, 705-708; Ekanger, R., Sand, T. E., ~greid, D., Christoffersen, T. & Deskeland, S.O. (2985} J. Biol.
em. 260, 3393-340i}.
cAMP-Depeadeat Proteia Kiaase Assays. After two washes with Dulbecco~s phosphate-buffered saline, cell pellets (2 x 106 cells } were lysed~in 0.5 ml of 20 mM
Tris (pH 7.5}, 0.1 mM sodium EDTA, 1 mM
dithiothreitol, 0.1 mM pepstatin, 0.1 mM antipain, 0.1 mM chymostatin, 0.2 mM leupeptin, 0.4 mg/nnl aprotinin, and o.5 mg/ml soybean trypsin inhibitor, using 100 strokes of a bounce homogenizes. After centrifugation (Eppendorf 5412} for 8 min,. the_supernatants were adjusted to 0.7 mg protein/ml and~assayed (Uhler, M.
~D. & McKnight, G. S. )1987} 3. Biol. Chem. ~6?, 15202 -15207) immediately. Assays (40 ~1 total volume} were performed for 10 min at 30°C and contained 200 uM ATP, 2.7 x 106 cpm y[32P]ATP, 20 mM MgCl2, 100 ACM Kemptide {Sigma K-1127) (Kemp, g. E., Graves, D. J., Benjamin, E. & Krebs, E. G. (1977) J. Biol. Chern. 252, 4888-4894) , 40 mM_ Tris (pR 7.5) , ~ 100 EcM protein kinase inhibitor (Sigma P-3294] (Cheng, H.-C.; Van Patten, S.
M., Smith, A. J. & Welsh, D. A.~ (1985) Biochem. J.
231, 655-661j , -!- 8 ACM cAMP and 7 ~a9 of cell extract.
The phosphorylation' of 'Kemptide was determined by spotting . 20 ~C1 of incubation mixture on phosphocellulose filters (Whatman, P81) and washing in phosphoric acid as described (Raskoski, R. (1983) Methods Enzvmol. ~, 3-6). Radioactivity was measured by liguid scintillation using Econofluor-2 (NEN
Research Products NEF-969). .__ Isolation of Total RNA and Northern Blot Analysis. The cells (108 washed twice with phosphate-buffered saline) Were lysed in 4.2 M guanidine isothiocyanate containing 25 mM sodium citrate (pFI
7.0), 0.5% sarcosyl (N-lauroylsarcosine Nat), and 0.1 M ~-mercaptoethanol, and the lysates were homogenized, and total cellular RNA was sedimented through a CsCl cushion (5.7 M CsCl, 10'mM EDTA) as described by Chirgwin et at. (Chirgwin, J. M., Przybyla, A. E., MacDonald, R.~Y. & Ratter, W. J. (1977) Biochemistry 18, 5284-5288). Total cellular RNA containing 2f mM
FIELD OF THE INVENTION
The invention is in the field of medicinal chemistry. In particular, the invention relates to certain antisense oligonucleotides and the use thereof for the treatment of cancer.
BACKGROUND OF THE INTENTION
Control mechanisms for cell growth and differentiation are disrupted in neoplastic calls (Potter, V.R. (1988) Adv.~Oncol. 4, 1-8; Strife, A. &
Clarkson, B. (1988) Semin. Hematol. 25, 1-19; Sachs, L. (1987) Cancer Res. 47, 1981-1986). cAMP, an~
intracellular regulatory agent, has been considered to have a role in the control of cell proliferation and differentiation (Pastan, I., Johnson, G.S. & Anderson, W.B. (1975) nn. Rev. Biochem. 44, 491-522; Prasad, K.N. (1975) Biol. Rev. 50, 129-165; ~Cho-Chung, X.S.
(1980) J. Cyclic Nucleotide Res. 6, 163°-177; Puck, T.T. (1987) Somatic Cell Mot. Genet._ 13, 451-457).
Either inhibitory or stimulatory effects of cAIKP on cell growth have been reported previously in studies in which cAMP analogs such as N6-OZ ~-dibutyryladenosine 3',5~-cyclic monophosphate or agents that raise ... 2 _ intracellular cAMP to abno.rznal and continuously high levels were used, and available data are interpreted.
very 'differently (chapowski, F.J., Kelly, L.A. &
Butcher, R.W. (1975) Adv. Cyclic Nucleotide Protein Phosphorylat. Res: 6, 245-338; Cho-Chung, Y.S. (1979) in Influence of Hormones on Tumor Development, eds.
Kellen, J.A. & Hilf, R. (CRC, Boca Raton, FL), pp. 55-93); Prasad, K.N. (1981) in The Transformed Cell, eds.
Cameron, L.L. & Pooh, T.B. (Academic, New York), pp.
235-266; Boynton, A.L. & Whit~ield, J.F. (1983) Aav.
Cyclic Nucleotide Res 15, 193-294).
Recently, site-selective CAMP analogs were discovered which show a preference for binding. to purified preparations of type II rather than type-' I
cAMP-dependent protein kinase in vitro (Robinson-Steiner, A.M. & Corbin, J.D. (1983) J. Biol. Chem.
258, 1032-1040; f6greid, D., Ekanger, R., Suva, R.K., Miller, J.P., Sturm, P., corbin, J. D. & Deskeland, S.O. (1985) Eur. J. Biochem. 150, 2i9-227), provoke potent growth inhibition, differentiation,'and reverse transformation in a broad spectrum of human and rodent cancer cell lines (Katsaros, D., Tortora, G., Tagliaferri, P., Clair, T., Ally, S., Neckers, L., Robins, R.K. & Cho-Chung, Y.S. (1987) FEBS Lett. 223, 97=103; Tortora, G_, Tagliaferrz, P., Clair, T., Colamonici, O., Neckers, L_M., Robins, R.K. & Cho-Chung, Y..S. (1988) B oo , 71, 230-233; Tagliaferri "
F., Katsaros, D., Clair, T., Robins,. R.K. & Cho-Chung, Y.S. (1988) J. Biol. Chem. 263, 409-416). The type I
and type II protein kinases are distinguished by their regulatory subunits (RI and RII, respectively) (Corbin, J.D., Keely, S.L. & Park, C.R. (1975) 3.
Biol. C~em. 250,- 218-225; Ho~mann, F., Beavo, J.A. &
Krebs, E.G. (1975) J. Biol. Chew. 250, 7795-7801).
3-5 Four different regulatory subunits.[RI« (previously designated RI) (Lee, D.C., Carmichael, D.F., Krebs, E.G. & McXnight, G.S. (1983) Proc. Natl. Acad. Sci.
USA 8a, 3608-3612y, RTE (Clegg, C.i., Cadd, G.G.
&
McKnight, G.S. (i988j Proc. Natl. Aced. Sci. USA
85, 3703-3707), RTxa (RTT~y) (Scott, J.D., Glaccum, M.B., Zoller, M.J., Uhler, M.D., Hofmann, D.M., McXnight, G.S. & Krebs, E.G. (1987) Proc Natl Agad Sci LISA
84, 5192-5195) and ,RII~ (RII51) (Jahnsen, T., Iiedin, h., Kidd, V.J., Beattie, W.G., Lohmann, S.M., Walter, U., Durica, J., Schul2, T.Z., Schlitz, E., Browner, M., Irawrence, C,B:; Goldman, D., Ratoosh, S.k.
&
Richards, J.S. {1986) J. Biol. Chem. 261, 12352-12361)] have now been identified at the gene/mRNA
' level. Two different catalytic subunits [Ca (Lmler, M.D., Carmichael, D.F., Lee, D.C. Chrivia, J.C., Krebs, F.G. & McKnight, G.S. {x986) Proc. Natl.
Aced.
Sci. USA 83, 1300-1304) and C~ (Uhler, M.D,., Chrivia, .
261, J,C. ~& McKnight, G.S. (1986) ~T. Biol~, Chem.
15360-15363; Showers, M,o. & Maurer, R.A. (1986) J.
Siol. Chem. 261, 16288-16291)3 have also been identified; however, preferential coexpression of ~ either one of these catalytic subunits with either the type I or type II protein kinase regulatory subunit has not been found (Showers, M.o. & Maurer, R.A.
(1986) J. Biol. Chew: ~6x, 16288-16291).
The growth inhibition by site-selective cAl~
anatlogs parallels reduction in RIa with an increase in RII~, resulting in an increase of the RII~/Rx ratio in cancer cells (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros,, D., f~greid, D., D6skelarid, S.O., Jahnsen, T, & Cho-Chung, Y.S.
(1988) ~oc_ Natl. Aced. Sci. USA $~, 6319-6322; Gho-Chung, Y.S. (1989) J. Natl. Cancer Inst. 81, 982-987).
Such selection modulation of RIa versus RII~ is riot mimicked by ~:reatment with N6,02~-dibutyryladeno-sine 3~,5~-cyclic monophosphate, a,previously studied a 38 CAMP analog (Ally, S., Tortara, G.,~
Clair, 2~., Grieco, D. , Merlo, G. , ICatsaros, D. , Qlgreid, D. , Deskel:and, S.O., Jahnsen, T. & Cho-Chung, Y.S. (1.988) .Prop.
Nat7.w, ... 4 _ Aced. Sci. USA 85, 6319-6322). The growth inhibitian further correlates with a rapid translocation of RII~
to the nucleus and an increase in the transcription of the RII~ gene (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros, D., ggreid, D., Dgskeland, S.O., Jahnsen, T. & Cho-Chung, Y.S.
(1988) proc. Natl. Aced. Sci. USA 85,-6319-6322). These results support the hypothesis that RII~ plays an important role in the cAMP growth regulatory function {Cho--Chung, Y.S. (1989j J. Natl. Cancer Inst.
81, 982-987).
Antisense RNA sequences have been described as naturally occurring biological inhibitors of gene expression in both prokaryates (Mizuno, T., Chou, M-Y, arid Inouye, M. {1984), Proc. Natl. Aced. Sci.
USA 81, (1966-1970)) and eukaryotes (Heywood, S.M. Nucleic Acids Res., 14, 6771-67?2 (2986)), and these sequences presumably function by hybridizing to complementaz-y mRNA sequences, resulting in hybridization arrest of translation (Peterson, B.M., Roberts, B.E., and ICuff, E.L., {1977) Proc. Natl. Aced. Sci. USA, ?4, 4374. Antisense oligodeoxynucleotides are short synthetic nucleotide seguences formulated to be complementary to a specific gene or RNA message.
.
Through the binding of these oligomers to a target DNA
or mRNA sequence, transcription or translation of the gene carr be selectively blocked and the disease process generated by that gene can be halted.
The cytoplasmic location of mRNA provides a target 38 considered to be readily accessible to antisense oligodeoxynucleotides entering the cell; hence much of the work in the field has focused on RNA as a target.
Currently, the use of antisense oligodeoxynucleotides provides a useful tool for exploring regulation of .35 gene expression in vitro and in tissue culture (Rothenberg, M., Johnson, G.; Laughlin, C., Green, I., Craddock, d., Sarver, N., and Cohere, 3'.S.(1989j J.
Natl. Cancer Inst., 83:1539--1544.
SUhQ2ARY OF TFiE INVEPi'PION
The invention is related to the discovery that inhibiting the expression of RIa in leukemia cells by contact with an antisense o-oligonucleotides and S-oligonucleotides fo~'~RI results in the inhibition of proliferation and the stimulation of cell differentiation. Accordingly, the invention is directed to RIa antisense oligonucleotides and phar~aaceutical compositions thereof for the treatment of cancer.
In particular, the invention is related to 15-to 3o-mer antisense oligonucleotides which ~. are complementary to a region in the first 1,00 N-terminal codons of RIa (Seq. ID No:6j.
The invention is also related to 15- to 3o-mer antisense oligonucleotides which are a fragment of antisense DNA complementary to RI (Seq. ID No:
5j.
The invention is also related to pharmaceutical compositions comprising at least one 15- to 30-mer antisense oligonucleotide which is complementary to a region in the first 10o N-terminal codons of RIa (Seq.
ID No:6); and a pharmaceutically acceptable carrier.
The~invention is also related to a method for treating cancer by suppressing growth of cancer cells susceptible to growth suppression and for inducing cancer cell differentiation in an animal comprising administering to an animal in need of such treatment 3o a cancer cell growth suppressing amount of an RIa antisense oligonucleotide.
DESCRIPTIO~1 of ,~E F3,C~,URES
Fiq. 1 depicts a graph showing the effect of RId antisense oligodeoxynuclentide on the basal rata of growth of HL-60 leukemia cells {A} and the growth of these cells when treated with cAM.P analogs or TPA (B}.
A, cells were,grown {sae the Examples} in the absence (o} or presence (~} of RIa antisense oligodeoxy-nucleotide (15 ACM}. At indicated'~times, cell counts in duplicate were performed. Data represent the average values -r SD of four experiments: I3, on day ~
of experiment A, cells exposed or unexposed to RIB
antisense oligodeoxynucleotide were reseeded (day 0}
at 5 x los cellsJdish, and cells pre-exposed to RIQ
antisense oligodeoxynucleotide were further treated with the oligomer at day 0 and day 2. cAMp analogs and. TPA were added one time at day o. Cell counts were performed on a Coulter counter on day 4. 8-Cl, 8~C1-cAMP (10 ;.cM}; 8-C1 + N6-B, 8-C1-cAMP {5 ACM} + N6-benzyl-cAMP (5 ~M}; TPA (3.0 ~ M}_ The data represent the average values ~ SD of four experiments.
Fia. 2 depicts a graph showing the effect of RIa antisense oligodeoxynucleotide on the morphologic transformation of HL-60 cells. Cells either exposed or unexposed to RIa antisense oligodeoxynucleotide were treated with cAMP analogs or TPA as described in Fig. 1B. On day 4 (see Fig. 1B}, cells were washed twice in Dulbecco's phosphate-buffered saline and were pelleted onto a glass slide by cytocentrifuge. The resulting cytopreparations were fixed and stained by Wright's stain. x Z80.
Fia~s depicts a Northern blot showing decreased RIQ
mRNA expression in HL-60 leukemia cells exposed to RIa antisense oligodeoxynucleotide. Cells were either exposed or unexposed to RI« antisense oligadeoxynucleotide (15 ACM} for 8 hr. Isolation of total RNA and Northern plot analysis followed the methods described in the Examples. A, ethidium bromide staining of RNA; M, markers of ribosomal RNAs;
lanes l, 2, cells une~tposed or exposed to RIa antisense oligomer. .8, Northern blot analysis; the same nitrocellulose filter eaas hybridized to both RIa and actin probes in sequential manner: .Lanes Z, 2, cells unexposed or exposed to RIB antisense oligomer.
Fiq: 4 depicts an SDS-PAGE showing the~effect of RIa antisense oligodeoxynucleotide on the basal and induced levels of RI and RII~ cAMP receptor proteins in HL-60 leukemic cells. Cells were either exposed to RIa antisense oligodeoxynucleotide (15 ~CMj or treated with cAMP analogs as described in Fig. 1. Preparation of cell extracts, the photoactivated incorporation of g-N3-~32P]CAMP and immunoprecipitation using the anti-RIa or anti-RII~ antiserum and protein A Sepharose, and SDS-PAGE of solubilized antigen-antibody complex followed the methods described in the Examples.
Pre-immune serum controls were carried out simultaneously and detected no immunoprecipitated band. M, labeled marker proteins of known molecular weight;
RIQ, the 48,000 molecular weight RI (Sigma);
RIIa, the 56,000 molecular weight RII (Sigma). Lanes RIa and RII~ are from photoaffinity labeling with 8-N3-[32P]CAMP only; lanes 1 to 3, photoaffinity labeling with 8-N3-[s2P]CAMP followed by immunoprecipitation with anti-RI or anti-RII~ antiserum.. 8-C1, 8-Cl-cAMP
(5 ;CMj; N6-benzyl,Nfi-benzyl-eAMP (5~CMj. The data in the table represent quantification by densitometric scanning of the autoradiograms. The data are expressed relative to the levels in control cells unexposed to RI~~antisense oligomer and untreated with cAMP analog, which are set equal to_1 arbitrary unit_ ' The data represent an average SD of three a5 experiments. A and B, immunoprecipitation with anti-RIa and anti.-RII~ antisera, respectively.
g Ficx. 5 depicts graphs showing the growth inhibition of human cancer cell lines by RIQ antisense oligodeoxynucleotide having SEQ 7D No: 1 (O-oligo and S-oligo derivatives), compared to controls. Cell lines: SK-N-SH, neuroblastoma; T~S-174T, colon carcinoma; MCF-7, breast carcinoma; TMK-l, gastric carcinoma. E2, estradiol-17~.
Fict. s depicts the -change in morphology of 'sK-N-SA
human neuroblastoma sells exposed to RIQ antisense 1.0 oligodeoxynucleotide having SEQ ID No: 1..
Fiq. 7 depicts a graph showing that RIa antisense oligadeoxynucleotide and its phosphorothioate analog inhibit the in vivo growth of LS-174T human. colon carcinoma in athymic mice. Figure 7A shows. the 1.5 oligodeoxynucleotide concentration-dependent inhibition of tumor growth. a-oligo, RI« antisense oligodeoxynucleotide~ S-oligo, phosphorothioate analog of RIa antisense oligomer. The cholesterol pellets (total weight 20 mg) containing the indicated doses of 20 O-oligo or S-oligo were implanted s.c. one time, at zero time, and tumor sizes were measured. Tumor volume (see Materials and Methods, Example 3j represents an average ~ S.D. of 7 tumors. . Figure 7B
shows the temporal effect of antisense 25 oligodeoxynucleotide phosphorothioate analogs on tumor growth. S-oligos as indicated at 0.3 mg dose in cholesterol pellets (total weight 20 mg) were implanted s.c. 2x/week, and tumor volume (see Materials and Methads, Example 3) represents an 30 average 1- S.D. of 7 tumors.
-g-DESCRIPTION OF THE PREFERRED EMBODIMENTS
Antisense therapy is the administration of exogenous oligonucleotides which bind to a target polynucleotide located within the cells. The term "antisense" refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g. RI«. See for example, Jack Cohen, OLIGODEOXYNUCLEOTIDES, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The RIa antisense oligonucleotides of the present invention include derivatives such as S-aligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, s-upra) which exhibit enhanced cancer cell growth inhibitory action (see Figures 5 and 7A).
S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention may be prepared by treatment of the corresponding 0-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide which is a sulfur transfer reagent.
See Iyer, R.P. et al., J. Org. Chem. 55: 4693-4698 (1990); and Iyer, R.P. et al., J. Am. Chem. Soc.
112:1253-1254 (1990).
The RIa antisense oligonucleotides of the present invention may be RNA or DNA which is complementary to and stably hybridizes with the first 100 N-terminal codons of the RIa genome or the corresponding mRNA. Use of an oligonucleotide complementary to this region allows for the selective hybridization to RIa mRNA and not to mRNA specifying other regulatory subunits of protein kinase.
Preferably, the RIa antisense oligonucleotides of the present invention are a 15 to 30-mer fragment of the antisense DNA molecule having SEQ ID N0:5 which hybridizes to RIa mRNA.
Alternatively, RIa antisense oligonucleotide is a 15-to 30-mer oiigonucleotide which is complementary to a region in the first 100 N-terminal codons of RI« (Seq.
ID No:6). Most preferably, the RI« antisense oligonucleotide has SEQ ID No: 1, SEQ ID No: 2, SEQ
ID No: 3, or SEQ ID No: 4.
Included as well in the present invention are pharmaceutical compositions comprising an effective amount of at least one of the RT« antisense oligonucleotides of the invention in combination with a pharmaceutically acceptable carrier. In one embodiment, a single RIa antisense oligonucleotide is utilized. In another embodiment, two RIa antisense oligonucleotides are utilized which are complementary to adjacent regions of the RIa genome. Administration of two RIa antisense oligonucleotides which are complementary to adjacent regions of the RIa genome or v corresponding mRNA may allow for more efficient inhibition of RIa genomic transcription or mRNA
translation, resulting in more effective inhibition of cancer cell growth.
Preferably, the RIa antisense oligonucleotides is coadministered with an agent which enhances the uptake of the antisense molecule by the cells. For example, the RI« antisense oligonucleotide may be combined with a lipophilic cationic compound which may be in the form of liposomes. The use of liposomes to introduce nucleotides into cells is taught, for example, in U.S. Patent Nos. 4,897,355 and 4,394,448. See also U.S. Patent Nos. 4,235,871, 4,231,877, 4,224,179, 4,753,788, 4,673,567, 4,247,411, 4,814,270 for general methods of preparing liposomes comprising biological materials.
Alternatively, the RI« antisense oligonucleotide may be combined with a lipophilic carrier such as any one of a number o~ sterals including cholesterol, cholate and deoxycholic acid. A preferred sterol is cholesterol.
In addition, the RIa antisense oligonucleotide may be conjugated to a peptide that is ingested by cells. Examples of useful peptides include peptide .
and peptide toxins.
hormones, antigens or antibodies, By choosing a peptide that is selectively taken up by the neoplastic cells, specific delivery of the antisense .agent may be effected. The Rl~ antisense oligonucleotide may be covalently bound via the 5'OH.
group by formation of an activated aminoalkyl derivative. The peptide of choice may then be aovalen~ly attached to the activated RIa antisense al'igonucleotide via an amino and sulfhydryl reactive hetero bifunctional reagent. The latter is hound to a cysteine residue present in the peptide_ upon exposure of cells to the RIa antisense oligonucleotide bound to the peptide, the peptidyl. antisense agent is endocytosed and the RIa antisense oligonucleotide binds to the target RTa mRNA to inhibit translation.
See PCT Application Publication No. PCT/US89/02363.
As antineoplastic agents, the RIa antisense 0ligonucleotides of the present invention.are useful .
in txeating a variety of cancers, including, but not limited ta, gastric, pancreatic, lung, breast, anal, colorectal, head and neck neoplasms, neurablastomas, melanoma and various leukemias. ' The RIa antisense oligonucleotides of the invention may also be active against the following tumor systems: F9 teratocarcinoma, SK-N-SH
neuroblastoma, TMIC-1 gastric carcinoma, HL-60 promyelocytic I:eukemia, Leukemia L-3.210, Leukemia p388, P1534 leukemia, Friend Virus:,Leukemia, Leukemia L4946, Mecca lymphosarcoma, Gardner lymphosarcoma, Ridgway Osteogenic sarcoma, sarcoma 180 (ascites), Wagner osteogenic sarcoma, Sarcoma T241, Lewis lung carcinoma, Carcinoma 755, CDSF, MCF-7 breast carcinoma, Colon 38, LS-7.7~1T colon carcinoma, Carcinoma 1025, Ehrlich carcinoma (ascites & solid}, Krubs 2 carcinoma (ascites), Bashford carcinoma 63, Adenacarcinoma E 0771, B16 Melanoma, Hardin-Passey melanoma, Gilama 26, Miyona adenocarcinoma, Walker carcinosarcoma 256, Flexner-Job3ing carcinoma, Jensen sarcoma, Iglesias ~,arcoma, Iglesias ovarian tumor, Murphy-stern lymphosarcoma, Yoshida sarcoma, Dunning leukemia, Rous chicken sarcoma, and Crabb hamster sarcoma.
The RIB antisense oligonucleotides and the pharmaceutical compositions of the present invention may be administered by any means that achieve their intended purpose. For example, administration may be by ' parenteral, subcutaneous, intravenous, intramuscular, intxa-peritoneal, or ~transdermal routes. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
Compositions within the scope of this invention include all compositions wherein the RIa antisense oligonucleotide is contained in an amount which is effective to achieve inhibition of proliferation and/or stimulate differentiation of the subject cancer cells. While-individual needs vary, determination of optimal ranges of effective amounts of each component is with the skill of the art. Typically, the RIa antisense aligonucleotide. may be administered to mammals, e.g. humans, at a dose of 0.005 to 1 mg/kg/day, or an equivalent amount of the pharmaceuti-cally acceptable salt thereof, per day of the body weight of the mammal being treated:.
25 In addition to administering the RIa antisense oligonucleotides as a raw chemical in solution, the RI« antisense oligonucleotides may be administered as part of a pharmaceutical prepaxation containing suitable pharmaceutically acceptable carriers comprising exoipients and auxiliaries. which facilitate processing of the RIB antisense oligonualeotxde into preparations which can be used pharmaceutically.
Suitable formulations y for parenteral administration include ac,~ueous solutions of the Rxa antisense oligonucie~t~.des in water-soluble form.
far example, water-soluble salts. In addition, to . suspensions of the active compounds as appropriate oily . inj ection suspensions ,, may .~ be adnninistexed.
suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oii, or ~ synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
a5 Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxyiaethyl cellulose, soxbitol, and/or dextran. optionally, the suspension may also captain stabilizers.
20 The antisense oligonucleetides of the present invention may be prepared according to any of the methods that are well known to those o ordinary skill.
in the art. Preferably, the antisense ~
phase olir~onuoleotides are prepared by so3:id 25 synthesis. See, Goodchild, J. , ~3ioeon-~uaate Chem's, ~,,:165-167 X1990) , for a review of the chemical synthesis of ~olagonucleotides.
Alternative7~y,~ the antisense oligonualeotides can be obtained from a number of companies which specialize 30 in the custom synthesis of oligonueleotides. .
Having now generally described this invention, c the same will, be vx~darstood by reference to an example which is provided herein for purposes of illustration only and ~is not intending to _be~ limited unless 35 otherwise specified.
- i4 -EXAMPLES
~xamt~le 1 Oligodeoxynucleotides. The 2i-mer oligodeoxynucleo-tides used in the present studies were synthesized at Midland Certified Reagent Co. (Midland, TX) and had the following sequences: human.-RIa .(Sandberg, M., Tasken, K., Oyen, o., Hansson, V. & Jahnsen, T. {1987) Biochem. Biophys. ,f. Res. Common. X49, 939-945) antisense, 5~-GGC-GGT-ACT-GCC--AGA-CTC-CAT-3 ~ (SSQ ID
No:l); human RII~g (Levy, F.O., oyen, O., Sandberg, M., Tasken, K., Eskild, W., Hansson, V. & Jahnsen, T.
(1988) t~1_ Endocrinol., 2, 1364-1373) antisense 5'-CGC-CGG-GAT-CTC-GAT-GCT-CAT-3' (SEQ ID N0:8);
human RIIa Oyen, O., Mykelbust, F., Scott, J.D., Hansson, V. & Jahnsen, T. (1989) FEES Lett. 246, 57-64) antisense 5'-CGG-GAT-CTG-GAT-GTG-GCT-CAT-3' (SEQ ID No:9); and the random sequence oligodeoxynucleotide was made of a mixture of all four nucleotides at every position.
Cell tirowth Experiment. Cells grown in suspension culture in RPM1 1640 medium supplemented with l0% heat-inactivated fetal bovine serum, penicillin (50 U/ml), streptomyc3.n (500 ~ag/atl.), and 1 mM;glutamine (Gibco, Grand Island, NY) were seeded at x 105 cells per dish. Oligodeoxynueleotides were added after seeding and every 48 hr thereafter. Ceil counts were perfonced on a. Coultsr counter. Cells unexposed or exposed to oligodeoxynucleotides for 4 days were reseeded (day O) at 5 x z05 cells/dish, and cells pre-exposed to the oligodeoxynucleotide were further treated with the oligomer at day 0 and day 2.
eAMp analogs (kindly provided by Dr. R.K. Robins, Nucleic Acid Research Institute, Costa Mesa, CA) or 12-O-tetradecanoylghorbol-i3-aceta~,e (TPA3 were added one time at day 0. Cell counts were performed on day 4.
- is -Immunoprecipitation of RIa and RZI~ cA,MP Receptor Proteins after Photoaffinity Labeling with 8-A3-t32p~c~,. Cell extracts were prepared at 0-4°C. The cell pellets (2 x 104 calls) , after two washes with PBS, were suspended in 0.5 ml buffer Ten (0.1 M NaCl, 5 mM MgCl2, 1$ Nonidet P-40, 0.5% Na deoxycholate, 2 KIU/ml bovine aprotinin, and 20 inM Tris-IiCl, pH 7. 4 j containing proteolysis inhibitors (Tortora, G., Clair, T. & Cho-Chung, Y.~~~S. (1990) Proc Natl Acad. Sci.
~ 87, 705-708}, vortex-mixed, passed through a 22-gauge needle l0 times, allawed to stand for 3o min at 4°C, and centrifuged at 750 x g for 20 min; the resulting supernatants were used as,cell lysates. The photoactivated incorporation of 8-N3-[32P]cAMP (60.0 Ci/mmol}, and the immunoprecipitation using the anti-RIa or anti-RII~ antiserwn (kindly provided by Dr. S _ O.
Dgskeland, Universitg of Bergen, Bergen, Norway) and protein A Sepharose~ and SDS-PAGE of solubilized antigen-antibody complex followed the method previously described (Tortora, G., Clair, T. & Cho-Chung, Y. S. (1990) Proc. Natl. Aced. Sci. USA 87, 705-708; Ekanger, R., Sand, T. E., ~greid, D., Christoffersen, T. & Deskeland, S.O. (2985} J. Biol.
em. 260, 3393-340i}.
cAMP-Depeadeat Proteia Kiaase Assays. After two washes with Dulbecco~s phosphate-buffered saline, cell pellets (2 x 106 cells } were lysed~in 0.5 ml of 20 mM
Tris (pH 7.5}, 0.1 mM sodium EDTA, 1 mM
dithiothreitol, 0.1 mM pepstatin, 0.1 mM antipain, 0.1 mM chymostatin, 0.2 mM leupeptin, 0.4 mg/nnl aprotinin, and o.5 mg/ml soybean trypsin inhibitor, using 100 strokes of a bounce homogenizes. After centrifugation (Eppendorf 5412} for 8 min,. the_supernatants were adjusted to 0.7 mg protein/ml and~assayed (Uhler, M.
~D. & McKnight, G. S. )1987} 3. Biol. Chem. ~6?, 15202 -15207) immediately. Assays (40 ~1 total volume} were performed for 10 min at 30°C and contained 200 uM ATP, 2.7 x 106 cpm y[32P]ATP, 20 mM MgCl2, 100 ACM Kemptide {Sigma K-1127) (Kemp, g. E., Graves, D. J., Benjamin, E. & Krebs, E. G. (1977) J. Biol. Chern. 252, 4888-4894) , 40 mM_ Tris (pR 7.5) , ~ 100 EcM protein kinase inhibitor (Sigma P-3294] (Cheng, H.-C.; Van Patten, S.
M., Smith, A. J. & Welsh, D. A.~ (1985) Biochem. J.
231, 655-661j , -!- 8 ACM cAMP and 7 ~a9 of cell extract.
The phosphorylation' of 'Kemptide was determined by spotting . 20 ~C1 of incubation mixture on phosphocellulose filters (Whatman, P81) and washing in phosphoric acid as described (Raskoski, R. (1983) Methods Enzvmol. ~, 3-6). Radioactivity was measured by liguid scintillation using Econofluor-2 (NEN
Research Products NEF-969). .__ Isolation of Total RNA and Northern Blot Analysis. The cells (108 washed twice with phosphate-buffered saline) Were lysed in 4.2 M guanidine isothiocyanate containing 25 mM sodium citrate (pFI
7.0), 0.5% sarcosyl (N-lauroylsarcosine Nat), and 0.1 M ~-mercaptoethanol, and the lysates were homogenized, and total cellular RNA was sedimented through a CsCl cushion (5.7 M CsCl, 10'mM EDTA) as described by Chirgwin et at. (Chirgwin, J. M., Przybyla, A. E., MacDonald, R.~Y. & Ratter, W. J. (1977) Biochemistry 18, 5284-5288). Total cellular RNA containing 2f mM
3-[N-morpholine]propane-sulfonic acid (pH 7.0), 50%
foraiamide, and 6% formaldehyde was denatured at 65°C
for 10 min arid electrophoresed through a denaturing 1.2% agarose-2.2 M formaldehyde gel. The gels Were then transferred to BiotransT"'nylon membranes (ICN
Biomedicalsj by 'the method of Thomas (Thomas, P. S.
(1980) Proc. Natl. Aced. Sc . USA 7T, 5201-5205) arid hybridized to the following two 32P-labeled nick-translated eDNA probes: 1.5 kilobase (kb) cDNA clone containing the entire coding region for the human cAI~SP-dependent protein kinase type I regulatory subunit, ItTQ (Sandberg, M., Tasken, K., Oyen, 0,, . Hansson, V. & Jahnsen, T. (1387) Bio~em. Biophys.~
$es. Commun. 249, 939--945) {kindly provided by Dr. T.
Jahnsen, Institute of Pathology, Rikshospitalet, aslo, Norway), and human ~ actin {Oncor p7000 actin).
$ESULTS
The RIB antisense oligodeoxynucleotide at 15 ACM
to concentration had immediate effects on the rate of proliferation of HL-60 cells. By 4-5 days in culture, while cells unexposed to RId antisense oligomer demonstrated an exponential rate of growth, cells exposed to the antisense oligomer exhibited a reduced ' growth rate and eventually stopped replicating {Fig.
lA). This inhibitory effect on cell proliferation persisted throughout the culture period. The growth inhibition was not due to cell killing; cells were over 90% viable after exposuze to RIa antisense oligomer {15 ftM) for 7 days as assessed by flow cytometry using forward and side scatter. RIa sense, RIIQ, or RII~ antisense, or a random secyuence oli~odeoxynucleoti,de had ~ no such growth .inhibitory ef f eat .
Cells unexposed or exposed to RIB antisense aligodeoxynucleotide far 4 days in culture were reseeded and examined for their response to treatanent with cAMP analogs or TPA. In sells unexposed to RIa antisense oligodeoxynucleotide, 8-C1-cA3~P (10 ~Mj produced 60% growth inhibition, and 80% growth inhibition was achieved by 8-Cl-cAMP~(5 ~M) plus N6-benzyl-cAMP (5- ~Si~3) {Fig. 1Bj {Tortara, G.
, Tagliaferri, P., Clair, 2., Colamon~.ci, O.
Neckers, b.
M., Robixts, R. R. & Cho-Chung, Y. S. {19$8) S ood 7~, 230-233j, and TPA {10"$ M) exhibited 60% growth inhibitian (Fig. 1B). In contrast, cells exposed to CA 02488792 1991-10-28 . .____.. _ - Z8 ' antisense oligodeoxynucleotide exhibited retarded growth (25% the rate of growth of cells unexposed to _ the antisense oligomerj and neither cAMP analogs nor TPA brought about further retardation of growth (Fig.
1B) .
KL~60 cells undergo a monocytic differentiation upon treatment with site-selective cAMP analogs. Cells either unexposed .pr exposed to RIa antisense oligodeoxynucleotide were examined for their morphology before and after treatment with cAMP
analogs. As shown in Fig. 2, in cells unexposed to AIa antisense oligomer, 8-Cl-cAMP plus N6-benzyl-cAME~
induced a monocytic morphologic change characterized by a decrease in nuclear-to-cytoplasm ratio, abundant ruffled and vacuolated -cytoplasm, and loss of nucleoli. Strikingly, the same morphologic change was induced when cells were exposed to RIQ antisense oligodeoxynucleotide (Fig. 2). Moreover, the morphologic changes induced by antisense oligomer were indistinguishable from that induced by TPA (Fig. 2).
To provide more evidence that the growth inhibition and monocytic differentiation induced in HL-60 cells exposed to the RIa antisense oligodeoxynucleotide ware due to an intracellular effect of the oligomer, the RIa mRNA level was determined. As shown '1n Fig. 3, 3.0 kb RIa mRNA
(Sandberg, M., Tasken, K., oyen, O., Hansson, V. &
Jahnsen, T. 0.987) Biochem. Bioghvs: ljes. Commun. 1~4~,, 939-945) was virtually undetectable in cells exposed for 8 hr to RIa antisense oligodeoxynucleotide (Fig.' 3B, lane 2), and the decrease in RIB mRNA was not due to a lower amount of total RNA as shown by the ethidium bromide staining (compare lane 2 with lane 1 of Fig. 3A). Conversely, an enhanced level of actin mRNA was detected in cells exposed to RIa antisense oligomer (Fig., 3B). Whether the increase in actin CA 02488792 1991-10-28 _._.
mRNA level represents changes in cytoskeletal structure is not known.
The levels of cAMF receptor proteins in these cells was then determined by immunoprecipitation using anti-RIa and anti-RII~ antisera (Tortora, G.
, Clair, T.
& Cho-Chung, Y. S. (1990) roc Natl Acad Sci.
USA
87, ?05-708; Ekanger, R., Sand, T. E., Ogreid, D., Christoffersen, T. &~,.Deskeland, 8Ø (1.985) J. ~$iol.
Chem. 260, 3393-3401) after photoaffinity labeling of these receptor proteins with 8-Ng-[3zP]CAMP.
In control cells, treatment with 8-C1-cAMP plus benzyl-cAMP brought about a ?0% reduction in RIa with a 3-fold increase in RII~, resulting in a 10-fold increase in the ratio of RII~jRIa (Fig. 4) (cho-chung,, X. S. (1989) J. North Cancer Inst. 81, 982-98?).
Exposure of these cells to RIa antisense oligodeoxynucleatide for 4 days brought about marked changes in both and RIq and RII~ levels; an 80%
reduction in RIB with a 5-fold increase in RII~p resulted in a 25-fold increase in the ratio of RII~q/RI~
compared with that in control cells (Fig. 4) . Siince growth inhibition and differentiation were appreciable after 3-4 days of exposure to RIa antisense oligomer, the.changingllevels of RIa and RIx~ proteins appears to 35 be an early event necessary for commitment to differentiation.
Data~in Fig. 4 showed that suppression of RIa by the antisense oligodeoxynucleotide~ brought about a compensatory increase in RTI~ level. Such coordinated expression of RI and RII without changes in the amount of C subunit has been shown previously (Hofman, F_, Hechtel, P. J. & Krebs, E: G. (19??) J. Biol.
Ghem.
1442-144?; Otten, A. D. & Mcknight, G. S. (1989) J. Biol. Chew. 264, 20255-20260)._ The increase in RII~ may he responsible for. the differentiation induced in these cells after exposure to RIa antisense oligodeoxynucleotide. The increase in RII~ mRNA
or RII~ protein level has been correlated With cAMP
analog-induced differentiation in K-562 chronic . myelocytic leukemic cells (Tortora, G., Clair, T., Katsaros, D., Ally, S., Colamonici, t5., Neckers, L.
M., Tagliaferri, P., Jahnsen, T., Robins, R. K. & Cho Chung, -Y. S. (1989] ~~oc Natl cad Sci USA 86, 2849-2852) and in erythroid differentiation of Friend erythrocytic leukemia cells (Schwartx, D, A. & Rubin, C. S. (1985) J. Bio~. Chem. 260, 6296~6303) . Tn a 10~ recent report (Tortora, G., Clair, T. & Cho--Chung, Y.
S. (1990) ~r,~c Natl z", Acad Sci 'USA 87, 705°708) , we have provided direct evidence that RII~ is essential for the cAMP-induced differentiation int HIS-6o cells.
HL-60 cells that Were exposed to RII~ ~~antisense oligodeoxynucleotide became refractory to treatment with cAI~ analogs and continued to grow.
The essential role of RII~ in differentiation of HL-60 cells was further demonstrated when these cells were exposed to both RIa and RII~ antisense oligodeoxynucleotides simultaneously. As shown in Table 1, RIa antisense oligodeoxynucleotide induced a marked increase in the expression of monocytic surface antigens [Leu 15 (handay, A., Gartland, L. & Clement, h. ,T. (1983) ~?. Immunol. ~ 13"x,, 2757-2761) and Leu M3 (Dimitriu-Bona, A., Burmester, G. R., Waters, S. 3. &
Winchester, R. 3. (1983) ~. Immunol. 130, 145-152)]
along with a decrease in markers related to the immature myelogenous cells [My9 (Ta7.le, M. A., Rao, P.
E., Westberg, Fs., Allegar, N., Makowski, M., Mittler, R. S. & Goldstein, G. (:L983) Cell. Immunol. 78, 83.;
Todd, R. F. III, Griffin, J. D., Ritz, J., Nadler, L.
M. Abrams, T. & Schlossman, S. F. (1981) Leuk. Res. 5, 491)]. These changes in surface marker expression were abolished when cells were exposed simultaneously , to both and RIB and r RII~ antisense oligodeoxynucleotides (Table, 1). RII« cAMP receptor was not detected in HL-60 cells (Cho-Chung, Y. S., zi _ Clair, T., Tagliaferri, P., Ally, S., Katsaros, D., Tortora, G., Neckers, L., Avery, T. L., Grabtree, G.
. W. & Robins, R. K. (1989) Cancer Invest. 7(2), 161 1T?), and RIIa antisense oligodeoxynucleotide showed no interference with the effects of RIa antisense oligomer {Table 1). , Cells exposed to both RT« and RII~ antisense oliqodeoxynucleotid~ were neither growth inhibited nor differentiated regardless of cAMP analog 1.0 treatment. We interpret these results to reflect the ' blockage of cAMP-dependent growth regulatory pathway.
Cells 'under these conditions are no longer cAMP
dependent but survive and proliferate probably through an alternate pathway. Thus, suppression of both RI«
1S and RTI~ gene expression led to an abnormal cellulax growth regulation.similar to that in mutant cell lines (Gottesman, M. M. (1980) Ce 22, 329-330), those that contain either deficient or defective regulatory subunits of cAMP-dependent protein kinase and are no 20 longer sensitive to cAMg stimulus.
our results demonstrated that cAMP transduces signals for dual controls, either positive or negative, on cell proliferation, depending on the availability of R.t~ or RII~ receptor proteins. The RI«
25 antisense oligodeoxynucleotide which brought about suppression of RIa along with enhancement of RII~
expression led to terminal differentiation of HL-60 leukemia With no sign of cytotoxicity.
It is unlikely that free .C subunit increase in 30 cells exposed to RIB antisense oligodeoxynucleotide.
was responsible for the differentiation, because cells exposed to RII~ antisense or both RIa and RII~
antisense oligodeoxynucleotides, conditions which also would produce free C subunit, continued to grow anc~
35 became refractory to cAMp stimulus. In order L,o directly verify this we measured phosphotransferase activity in cells that are exposed or unexposed to ti. .
antisense oligodeoxynucleotides using kemptide (Kemp, B. E., Graves, D. J., Benjamin, E. & Krebs, E. G.
(1977) J. Biol. Chem. 252, 4888-4894) as a substrate in the presence and absence of a saturating concentration of CAMP and in the presence and absence of the heat-stable protein kinase inhibitor (Cheng, H.-C., Van Patten, S. M., Smith, A. J. & Welsh, D. A.
(1985) $3.OChe7lt~J. .r~31,, 65$'-661) . This method Of assay gives accurate deterzaination of the relative l0 levels Of dissociated C and total C activity.
'Cell extracts from untreated HL-60 cells exhibited a very low level of dissociated C and were stimulated 36-fold by cAMP (Table 2j. This cAMP-stimulated activity was almost completely inhihited by the heat-stable protein kinase inhibitor (Table 2j, indicating that the total C activity measured was cAMP-dependent protein kinase.
Tn cells exposed to RIa antisense, RII~ antisense, or RIa and Rxx~ antisense oligodeoxynualeotide, the free C activity was not increased as compared to unexposed control cells, although there Was a small difference in the total cAMP-stimulated activity (Table 2).
These results provide direct evidence that free catalytic subunit is not responsible for the differentiation observed in HL-6o cells.
over expression of Rza cAMP receptor protein has also been found in the majority of human breast and colon primary carcinomas examined (Sradbury, A. W., Miller, W. R., Clair, fi., Yokozaki,~H. & Ch0-Chung, Y.
S. (1990) Proc. Am. Assoc. Cancer Res. ~1, 172), suggesting an important .in vivo role of cAMP
receptor in tumor growth as well. However, the precise role of RIa in cell proliferation is not known at present.
RIa may suppress RIIS production by titxating out C
subunit, Or it may be a transducer of mitogeni.r.
signals leading to cell proliferation. our result-:.:
demonstrate that RIa antisense oligodeoxynucleotide provides a useful genetic tool for studies on the ro:~
- z3 -of cAMP receptor proteins in cell proliferation and differentiation, and contribute to a new approach in the control of malignancy.
Table I. Modulation of differentiation markers in HL-S 60 cells by RIa antisense oligadeoxynucleotide Surface Makers Treatment Leul5 LeuM3 My9 Control 10 2 lU0 RIQ.antisense 80 98 80 RIa antisense + RII~ antisense 11 2 _ 7.00 "
RIZ~ antisense 13 3 100 RIa antisense + RTIa antisense 85 loo 80 Surface antigen analysis was performed by flow cytometry using monoclonal antibodies reactive with either monocytic or myeloid cells. The monoclonal antibodies used were Leu 15, Leu M3, and My9. 2 x 104 cells were analyzed for each sample, and cell gating was~performed using forward and side scatter. The " numbers represent % positive and represent the average values of'three experiments.
Table 2. Protein kinase activity in HI.-GO cells Treatment Activity Relative Activity Relative 5tiauiatton -GAlIP to control e.cAHp to cmtro.t (foldD
- HKI
Controt 23.0 + 6,6 i.0 837 + 8T 1.0 35 Rtp antisesue 22.p f 5.4 1.0 944 t 18 1.1 41 RII~ anHse~e 22.8 f 8.4- 1.0 1,028 154 . 1:2 IO RIO snd RII~ aniisets~e 24.3 ; 7.0 1,1 802 ~ 36 1.0 33 ~ ?XI
Ca~trol 17.5 + 8.7 1.0 37.0 a 8.4 Z.i 1.0 Ria antise~e 25.0 r 8.8 1.4 22.6 f 8.8 0.9 0.6 RIt~ antismse 24.0 ; 2.6 1,4 26.8 3.9 0.7 i.0 REQ atd Rti~ antisense 19.0 ~ 5.9 1.1 tp.t + 8.2 1.0 0.5 Cells were.exposed to each of 15 ~uM concentrations of RIB, RII~r or RIa and RII~ antisense oligodeoxynucleo-tide for 4 days as shown in fig. 1A. fihe data represent an average ~ SD of duplicate determinations of three identical experiments.
*Picomoles phosphate transferred to ICemptide per min/mg protein.
Example 2 Next, the RIa antisense oligonucleotide having SEQ ID NO:l was administered to mice having an experimental tumor. A pellet of RI« antisense oligonucleotide (25 mgiKg) and cholesterol (1000 mg/Kg) was implanted s.c. in the left flank of athymic mice which had been injected in the right flank with 'LS-174T human colon cancer cells (2x106 cells) suspended in phosphate-buffered='saline. Tumor measurements and mouse weights were recorded on the initial day of treatment 4staging day), and at the end of treatment (staging day +5). the mean tumor weight zg change {D), was based on length and width measurements in millimeters. After a few days, the tumor growth was inhibited when compared to control cells (see Table 3}. No change in body weight was noted in the control and treated animals.
Table 3. Effect of RIa antisense oiigodeoxynucleo-tide s.c, pellet ow the growth of IsS-174T human colon carcinoma in athymic mice intci~t o~~, Fsm ~~,d x cucao~ ut tag) tuno~ wt tm9) at~ac~
Treatments s.c. pellet implanted Control 25 450 --RIa antisense 25 230 48.
(0.5 mg) 8--C1 cAMP {1 mg}b34 250 51 Nsbenzyl cAMP (1 mg) a. 20 mg pellet lyophilized consisting of indicated doses of RIa antisense or cAMP analogs plus supplement doses of cholesterol.
b. The growth inhibitory effect of these cAMP
analogs correlate with decrease in RIa (Natl_._, Cancer Inst. 81 982 {198_9}) and is shown here for comparison.
c. Mean tumor weight per group (4 mice) on staging_day.
d. Mean tumor weight per group on staging day +5.
e. % of change in test tumor weight (aT)/change in control tumor weight (~C).
In other in vitro experiments, the RI« antisense oligonucleotide having SEQ ID NO: 1 was added to dishes containing neuroblastoma,=''colon carcinonea, breast carcinoma and gastric carcinoma cells. A;:
shown in Figure 5, the RIa antisense oligonucleotide having SEQ ID No: 1, inhibited proliferation of uj' cancer cell types When compared to control cells.
Moreover, the Rra antisense oligonucleotide having SEQ
. ID No: 1 caused differentiatioai of the human neuroblastoma cells (see Figure 6).
Examaple 3 -Next, the effect of O-oligo and S-oligo RIB
antisanse oligonucleotides an the growth of LS-174T
human colon carcinoma in athymic mice was compared.
Materials and Methods. We synthesized_[Milligen Biosearch 8700 DNA synthesizer (Bedford, MA)] the 21-mer antisense oligodeoxynucleotides and their phosphorothioate analogs complementary to the human RIa, human RII~ mRIIA transcripts starting from the first colon, and mismatched sequence (random) oligomers of identical sire. The aligomers had the following sequences: RIa antisense, 5~-GGC-GG~°-ACT-GCC-AGA-CTC-CAT-3 ~ (SEQ ID No: I); Fttl~ antisense, i'-CGC-CGG-GAT-CTC-GAT-GCT-CAT-3' (SEQ ID No: 8~i and random oligo, 5'-CGA-TCG-ATC-GAT-CGA-TCG-TAC-3 ~ (SEQ ID No: 7).
LS-174T human colon carcinoma cells (2 x 106) Were injected s.c. in athymic mice, and the antisense oligodeoxynucleotides in the form of either a cholesterol pellet or 50% sesame oil emulsion were administered s.c_ 1 week later when mean tumor sizes usually were 25-50 mg. Tumor volume was based on.
length and width measurements and calculated by the formula 4/3 rte, where r ~ (length~+ width)/4.
Results and Disaussioa: Fig. 7 shows the dose-and time-dependent affect of an RIa antisense oligodeoxynucleotide (o--oligo) at 0.2 and 0.5 mq doses in cholesterol pellets administered s.c. one time (at zero time); it' brought about. 20 and 46% growth inhibition, respectively, in 7 days, When compared with control (untreated) tumors (Fig. 7A). Strikingly, f~he RIa antisense phosphorothioate analog (S-oligo) at a 0.2 mg dose (cholesterol pelT.et, s.c.) gave a 60%
growth inhibition at day 7, exhibiting a 3-fold greater potency than the 0-oliga antisense (Fig. 7A).
The growth inhibitory effect of RIa antisense S-oligo was even greater when animals were treated for a longer period: The Rla antisense S-oligo at a 0.3 mg dose in a cholesterol pellet, ~2 timesJweek s.c.
implantation for 3 weeks, resulted in a 80% growth inhibition; the tumor growth almost stopped after 2 weeks of treatment (Fig. ?B). RIa antisense O-oligo ZO or S-oligo administered s.c. as 50% sesame oil emulsion gave similar results. RIa antisense S-oligo brought ahout no apparent toxicity in animals; no body weight loss or other toxic symptoms were observed during the 3 weeks of treatment.
' The growth inhibitory effect brought about_by RIa antisense S-oligo was the specific effect of the oligomer: RII~ antisense or random (mismatched sequence) S-oligos of the identical size as the RIa antisense oligomer had no effect on the tumor growth (Fig. ?B).
To provide more evidence that the growth inhibition observed in colon carcinomas in athymic mice treated with RIa antisense oligodeoxynucleotide was due to an intracellular effect of the oligomer, the levels of Rh and RII~ cAMP receptor proteins in these tumors were determined. RIa levels were determined~by immunoblotting {Ally, S., Proc. Hall.
cad. Sci. USA 85:6319-5322 (1988)) using monoclonal antibody against human Rla (kindly provided by Drs.
T. I~ea, University of Oslo, Oslo, Norway, and S.O. Deskeland, University of Bergen, Bergen, Norway), and RII~ was measured by immunoprecipitation (Tortora;
G., et al., Proc. Natl. Aced. Sci. USA 87:705-?08 (1990)) with anti-RIT~ antiserum (kindly provided by Dr. S.t?. Deskeland) after ghotoaffinity lak~eling of RII~ with [32P]. 8-H3-cAMP. As shown in Table 4, RIa antisense s-oligomer treatment brought about a marked - zs -reduction (80% decrease) of RIB level in tumors as compared with that in untreated control tumors. This suppression of RIa expression by Rx« antisense S-oligomer brought about a 2-fold increase in RII~ level (Table 4 ) . Such coordinated expression of R:Ia and RThg without changes in the amount of catalytic subunit of protein kinase has been shown in HI.-60 leukemia cells that demonstrated growth inhibition and differentiation upon exposure to RIa antisense oligodeoxynucleotide. On the other hand, a 50%
increase in RIa level along with 80% suppression in RII~ level was observed in tumors after treatment with RII~g antisense S-oligomer (Table 4) which had no effect on tumor growth (Fig. 7). Random (mismatched 3.5 secquencej S-oligomer which had no effect on, tumor growth (Fig. 7) also showed no effect on RIa levels (Table 4j. Thus, reduction in RIa expression appears to trigger a decrease or halt in tumor growth upon treatment with RIa antisense oligomer. Our results demonstrated that cAMP transducer signals fox dual control, either positive or negative, on cell proliferation, depending on the availability of RI« or RII~ receptor proteins. The RIQ antisense oligodeoxynucleotide, which suppressed RIa and enhanced RII~ expression, led to inhibition of in vivo growth of solid colon carcinoma in athymic mice with no symptoms of toxicity in animals. The phosphorothioate analog (S-oligo). of RI,~ antisense oligomer exhibited a greater potency than the antisense of unmodified oligodeoxynucleotide (O-oligo). It has been shown that S-oligos, as compared with O-oligos, more readily enter cells, are more resistant to e:idonucleases, and yet exhibit high efficacy in hybridization with target mRNAs or DNAc (Stein, C.A., et al., In: J.S. Cohen (ed.jr Olirxodeoxynucleotides: ~ Antiser~se Inhibitors of Gene _ 29 Expression, pp. 97-117. Boca Raton, FL, CRC Press, Inc. (1989)).
These results demonstrate here for the first time the striking in vivo effect of antisense oligodeoxynucleotide in the suppression of, malignancy.
The depletion of RId, the type I regulatory subunit of cAMP-dependent protein kinase, by means of an antisense oligodeoxynucleotide, especially with its phosphorothioate analog,.leads to a successful halt of 30 tumor growth in vivo with no symptoms of toxicity, suggesting great potential of this antisense oligodeoxynucleotide fox clinical application.
Table 4 Suppression of RIa cAMP Receptor Expression by RI«
Antisense Oligodeoxynucleotide (s-oligo) Results in Compensatory Increase in RTIa Receptor Relative Levels Treatment RIB RII~
None 1.0 -1 0.1 1.0 ~ 0.1 RI« antisense S-oligo 0.2 -1 0.03 2.0 ~ 0.2 RIIB antisense S-oligo 1.5 ~ 0.2 0.2 ~ 0.02 Random S-~oligo 1.0 -i 0.1 1.0 ~ 0.1 Treatment with S-oligos as indicated were the same as that in Fig. 7B. At the end of the experiment (3 ~0 weeks), tumor extracts were prepared as previously described (Ally, S. et al., Cancer Res. 49:5650-5655 .(1980)) and immunoblotting and immunoprecipitation of RTa ,and RII~, respectively, were performed as previously described by Ally, S., et al., Proc. Natl.
Acad. Sci. USA 85:6319-6322 (1988) and Tortora, G., et al., Proc_ Natl. Ac~,d. Sci. USA 87:705-708_ (2990).
Data are from quantification by densitometric scanning of autoradiograms. Data are expressed relative to levels in control tumors (no treatment), which are set to equal to one as an arbitrary unit.
Data represent an average ~ S.D. of 7 tumors.
_ ~a In the following sequence listing, Seq ID No: 1 represents an antisense sequence corresponding to the first 7 N-terminal colons for RIa. Seq ID No: 2 represents an.antisense sequence corresponding to the 8th-13th colon for RI«. Seq ID No: 3~ represents an antisense sequence corresponding to the 14th-20th colon for RIa. Seq ID No: 4 represents an antisense ..~~ _ sequence corresponding to the 94th-100th colon for RIB.
Seq ID No: 5 represents an antisense sequence corresponding to the lgt-100th colon for RIa. Seq ID
No: 6 represents the sense sequence corresponding to the 18t-100th colon for RI«.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Yoon S. Cho-Chung (ii) TITLE OF INVENTION: Antisense Oligonucleotides for Treatment of Cancer (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MBM & CO.
(B) STREET: P.O. BOX 809, STATION B
(C) CITY: OTTAWA
(D) PROVINCE: ONTARIO
(E) COUNTRY: CANADA
(F) POSTAL CODE: K1P 5P9 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible {C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Ver. 2.1 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,054,325 (B) FILING DATE: 1991-10-28 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 607,113 (B) FILING DATE: 1990-11-02 {C) CLASSIFICATION:
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(A) APPLICATION NUMBER: US 702,163 (B) FILING DATE: 1991-05-20 {C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SWAIN, Margaret (B) REGISTRATION NUMBER: 10926 (C) REFERENCE/DOCKET NUMBER: 127-115 {ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 613/567-0762 (B) TELEFAX: 613/563-7671 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense DNA complementary to Protein Kinase subunit RIa mRNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
INFORMATTON FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo Sapiens (ix) FEATURE:
{A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Sequence: Partial sequence of Protein Kinase Regulatory Subunit RIa (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleio Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
foraiamide, and 6% formaldehyde was denatured at 65°C
for 10 min arid electrophoresed through a denaturing 1.2% agarose-2.2 M formaldehyde gel. The gels Were then transferred to BiotransT"'nylon membranes (ICN
Biomedicalsj by 'the method of Thomas (Thomas, P. S.
(1980) Proc. Natl. Aced. Sc . USA 7T, 5201-5205) arid hybridized to the following two 32P-labeled nick-translated eDNA probes: 1.5 kilobase (kb) cDNA clone containing the entire coding region for the human cAI~SP-dependent protein kinase type I regulatory subunit, ItTQ (Sandberg, M., Tasken, K., Oyen, 0,, . Hansson, V. & Jahnsen, T. (1387) Bio~em. Biophys.~
$es. Commun. 249, 939--945) {kindly provided by Dr. T.
Jahnsen, Institute of Pathology, Rikshospitalet, aslo, Norway), and human ~ actin {Oncor p7000 actin).
$ESULTS
The RIB antisense oligodeoxynucleotide at 15 ACM
to concentration had immediate effects on the rate of proliferation of HL-60 cells. By 4-5 days in culture, while cells unexposed to RId antisense oligomer demonstrated an exponential rate of growth, cells exposed to the antisense oligomer exhibited a reduced ' growth rate and eventually stopped replicating {Fig.
lA). This inhibitory effect on cell proliferation persisted throughout the culture period. The growth inhibition was not due to cell killing; cells were over 90% viable after exposuze to RIa antisense oligomer {15 ftM) for 7 days as assessed by flow cytometry using forward and side scatter. RIa sense, RIIQ, or RII~ antisense, or a random secyuence oli~odeoxynucleoti,de had ~ no such growth .inhibitory ef f eat .
Cells unexposed or exposed to RIB antisense aligodeoxynucleotide far 4 days in culture were reseeded and examined for their response to treatanent with cAMP analogs or TPA. In sells unexposed to RIa antisense oligodeoxynucleotide, 8-C1-cA3~P (10 ~Mj produced 60% growth inhibition, and 80% growth inhibition was achieved by 8-Cl-cAMP~(5 ~M) plus N6-benzyl-cAMP (5- ~Si~3) {Fig. 1Bj {Tortara, G.
, Tagliaferri, P., Clair, 2., Colamon~.ci, O.
Neckers, b.
M., Robixts, R. R. & Cho-Chung, Y. S. {19$8) S ood 7~, 230-233j, and TPA {10"$ M) exhibited 60% growth inhibitian (Fig. 1B). In contrast, cells exposed to CA 02488792 1991-10-28 . .____.. _ - Z8 ' antisense oligodeoxynucleotide exhibited retarded growth (25% the rate of growth of cells unexposed to _ the antisense oligomerj and neither cAMP analogs nor TPA brought about further retardation of growth (Fig.
1B) .
KL~60 cells undergo a monocytic differentiation upon treatment with site-selective cAMP analogs. Cells either unexposed .pr exposed to RIa antisense oligodeoxynucleotide were examined for their morphology before and after treatment with cAMP
analogs. As shown in Fig. 2, in cells unexposed to AIa antisense oligomer, 8-Cl-cAMP plus N6-benzyl-cAME~
induced a monocytic morphologic change characterized by a decrease in nuclear-to-cytoplasm ratio, abundant ruffled and vacuolated -cytoplasm, and loss of nucleoli. Strikingly, the same morphologic change was induced when cells were exposed to RIQ antisense oligodeoxynucleotide (Fig. 2). Moreover, the morphologic changes induced by antisense oligomer were indistinguishable from that induced by TPA (Fig. 2).
To provide more evidence that the growth inhibition and monocytic differentiation induced in HL-60 cells exposed to the RIa antisense oligodeoxynucleotide ware due to an intracellular effect of the oligomer, the RIa mRNA level was determined. As shown '1n Fig. 3, 3.0 kb RIa mRNA
(Sandberg, M., Tasken, K., oyen, O., Hansson, V. &
Jahnsen, T. 0.987) Biochem. Bioghvs: ljes. Commun. 1~4~,, 939-945) was virtually undetectable in cells exposed for 8 hr to RIa antisense oligodeoxynucleotide (Fig.' 3B, lane 2), and the decrease in RIB mRNA was not due to a lower amount of total RNA as shown by the ethidium bromide staining (compare lane 2 with lane 1 of Fig. 3A). Conversely, an enhanced level of actin mRNA was detected in cells exposed to RIa antisense oligomer (Fig., 3B). Whether the increase in actin CA 02488792 1991-10-28 _._.
mRNA level represents changes in cytoskeletal structure is not known.
The levels of cAMF receptor proteins in these cells was then determined by immunoprecipitation using anti-RIa and anti-RII~ antisera (Tortora, G.
, Clair, T.
& Cho-Chung, Y. S. (1990) roc Natl Acad Sci.
USA
87, ?05-708; Ekanger, R., Sand, T. E., Ogreid, D., Christoffersen, T. &~,.Deskeland, 8Ø (1.985) J. ~$iol.
Chem. 260, 3393-3401) after photoaffinity labeling of these receptor proteins with 8-Ng-[3zP]CAMP.
In control cells, treatment with 8-C1-cAMP plus benzyl-cAMP brought about a ?0% reduction in RIa with a 3-fold increase in RII~, resulting in a 10-fold increase in the ratio of RII~jRIa (Fig. 4) (cho-chung,, X. S. (1989) J. North Cancer Inst. 81, 982-98?).
Exposure of these cells to RIa antisense oligodeoxynucleatide for 4 days brought about marked changes in both and RIq and RII~ levels; an 80%
reduction in RIB with a 5-fold increase in RII~p resulted in a 25-fold increase in the ratio of RII~q/RI~
compared with that in control cells (Fig. 4) . Siince growth inhibition and differentiation were appreciable after 3-4 days of exposure to RIa antisense oligomer, the.changingllevels of RIa and RIx~ proteins appears to 35 be an early event necessary for commitment to differentiation.
Data~in Fig. 4 showed that suppression of RIa by the antisense oligodeoxynucleotide~ brought about a compensatory increase in RTI~ level. Such coordinated expression of RI and RII without changes in the amount of C subunit has been shown previously (Hofman, F_, Hechtel, P. J. & Krebs, E: G. (19??) J. Biol.
Ghem.
1442-144?; Otten, A. D. & Mcknight, G. S. (1989) J. Biol. Chew. 264, 20255-20260)._ The increase in RII~ may he responsible for. the differentiation induced in these cells after exposure to RIa antisense oligodeoxynucleotide. The increase in RII~ mRNA
or RII~ protein level has been correlated With cAMP
analog-induced differentiation in K-562 chronic . myelocytic leukemic cells (Tortora, G., Clair, T., Katsaros, D., Ally, S., Colamonici, t5., Neckers, L.
M., Tagliaferri, P., Jahnsen, T., Robins, R. K. & Cho Chung, -Y. S. (1989] ~~oc Natl cad Sci USA 86, 2849-2852) and in erythroid differentiation of Friend erythrocytic leukemia cells (Schwartx, D, A. & Rubin, C. S. (1985) J. Bio~. Chem. 260, 6296~6303) . Tn a 10~ recent report (Tortora, G., Clair, T. & Cho--Chung, Y.
S. (1990) ~r,~c Natl z", Acad Sci 'USA 87, 705°708) , we have provided direct evidence that RII~ is essential for the cAMP-induced differentiation int HIS-6o cells.
HL-60 cells that Were exposed to RII~ ~~antisense oligodeoxynucleotide became refractory to treatment with cAI~ analogs and continued to grow.
The essential role of RII~ in differentiation of HL-60 cells was further demonstrated when these cells were exposed to both RIa and RII~ antisense oligodeoxynucleotides simultaneously. As shown in Table 1, RIa antisense oligodeoxynucleotide induced a marked increase in the expression of monocytic surface antigens [Leu 15 (handay, A., Gartland, L. & Clement, h. ,T. (1983) ~?. Immunol. ~ 13"x,, 2757-2761) and Leu M3 (Dimitriu-Bona, A., Burmester, G. R., Waters, S. 3. &
Winchester, R. 3. (1983) ~. Immunol. 130, 145-152)]
along with a decrease in markers related to the immature myelogenous cells [My9 (Ta7.le, M. A., Rao, P.
E., Westberg, Fs., Allegar, N., Makowski, M., Mittler, R. S. & Goldstein, G. (:L983) Cell. Immunol. 78, 83.;
Todd, R. F. III, Griffin, J. D., Ritz, J., Nadler, L.
M. Abrams, T. & Schlossman, S. F. (1981) Leuk. Res. 5, 491)]. These changes in surface marker expression were abolished when cells were exposed simultaneously , to both and RIB and r RII~ antisense oligodeoxynucleotides (Table, 1). RII« cAMP receptor was not detected in HL-60 cells (Cho-Chung, Y. S., zi _ Clair, T., Tagliaferri, P., Ally, S., Katsaros, D., Tortora, G., Neckers, L., Avery, T. L., Grabtree, G.
. W. & Robins, R. K. (1989) Cancer Invest. 7(2), 161 1T?), and RIIa antisense oligodeoxynucleotide showed no interference with the effects of RIa antisense oligomer {Table 1). , Cells exposed to both RT« and RII~ antisense oliqodeoxynucleotid~ were neither growth inhibited nor differentiated regardless of cAMP analog 1.0 treatment. We interpret these results to reflect the ' blockage of cAMP-dependent growth regulatory pathway.
Cells 'under these conditions are no longer cAMP
dependent but survive and proliferate probably through an alternate pathway. Thus, suppression of both RI«
1S and RTI~ gene expression led to an abnormal cellulax growth regulation.similar to that in mutant cell lines (Gottesman, M. M. (1980) Ce 22, 329-330), those that contain either deficient or defective regulatory subunits of cAMP-dependent protein kinase and are no 20 longer sensitive to cAMg stimulus.
our results demonstrated that cAMP transduces signals for dual controls, either positive or negative, on cell proliferation, depending on the availability of R.t~ or RII~ receptor proteins. The RI«
25 antisense oligodeoxynucleotide which brought about suppression of RIa along with enhancement of RII~
expression led to terminal differentiation of HL-60 leukemia With no sign of cytotoxicity.
It is unlikely that free .C subunit increase in 30 cells exposed to RIB antisense oligodeoxynucleotide.
was responsible for the differentiation, because cells exposed to RII~ antisense or both RIa and RII~
antisense oligodeoxynucleotides, conditions which also would produce free C subunit, continued to grow anc~
35 became refractory to cAMp stimulus. In order L,o directly verify this we measured phosphotransferase activity in cells that are exposed or unexposed to ti. .
antisense oligodeoxynucleotides using kemptide (Kemp, B. E., Graves, D. J., Benjamin, E. & Krebs, E. G.
(1977) J. Biol. Chem. 252, 4888-4894) as a substrate in the presence and absence of a saturating concentration of CAMP and in the presence and absence of the heat-stable protein kinase inhibitor (Cheng, H.-C., Van Patten, S. M., Smith, A. J. & Welsh, D. A.
(1985) $3.OChe7lt~J. .r~31,, 65$'-661) . This method Of assay gives accurate deterzaination of the relative l0 levels Of dissociated C and total C activity.
'Cell extracts from untreated HL-60 cells exhibited a very low level of dissociated C and were stimulated 36-fold by cAMP (Table 2j. This cAMP-stimulated activity was almost completely inhihited by the heat-stable protein kinase inhibitor (Table 2j, indicating that the total C activity measured was cAMP-dependent protein kinase.
Tn cells exposed to RIa antisense, RII~ antisense, or RIa and Rxx~ antisense oligodeoxynualeotide, the free C activity was not increased as compared to unexposed control cells, although there Was a small difference in the total cAMP-stimulated activity (Table 2).
These results provide direct evidence that free catalytic subunit is not responsible for the differentiation observed in HL-6o cells.
over expression of Rza cAMP receptor protein has also been found in the majority of human breast and colon primary carcinomas examined (Sradbury, A. W., Miller, W. R., Clair, fi., Yokozaki,~H. & Ch0-Chung, Y.
S. (1990) Proc. Am. Assoc. Cancer Res. ~1, 172), suggesting an important .in vivo role of cAMP
receptor in tumor growth as well. However, the precise role of RIa in cell proliferation is not known at present.
RIa may suppress RIIS production by titxating out C
subunit, Or it may be a transducer of mitogeni.r.
signals leading to cell proliferation. our result-:.:
demonstrate that RIa antisense oligodeoxynucleotide provides a useful genetic tool for studies on the ro:~
- z3 -of cAMP receptor proteins in cell proliferation and differentiation, and contribute to a new approach in the control of malignancy.
Table I. Modulation of differentiation markers in HL-S 60 cells by RIa antisense oligadeoxynucleotide Surface Makers Treatment Leul5 LeuM3 My9 Control 10 2 lU0 RIQ.antisense 80 98 80 RIa antisense + RII~ antisense 11 2 _ 7.00 "
RIZ~ antisense 13 3 100 RIa antisense + RTIa antisense 85 loo 80 Surface antigen analysis was performed by flow cytometry using monoclonal antibodies reactive with either monocytic or myeloid cells. The monoclonal antibodies used were Leu 15, Leu M3, and My9. 2 x 104 cells were analyzed for each sample, and cell gating was~performed using forward and side scatter. The " numbers represent % positive and represent the average values of'three experiments.
Table 2. Protein kinase activity in HI.-GO cells Treatment Activity Relative Activity Relative 5tiauiatton -GAlIP to control e.cAHp to cmtro.t (foldD
- HKI
Controt 23.0 + 6,6 i.0 837 + 8T 1.0 35 Rtp antisesue 22.p f 5.4 1.0 944 t 18 1.1 41 RII~ anHse~e 22.8 f 8.4- 1.0 1,028 154 . 1:2 IO RIO snd RII~ aniisets~e 24.3 ; 7.0 1,1 802 ~ 36 1.0 33 ~ ?XI
Ca~trol 17.5 + 8.7 1.0 37.0 a 8.4 Z.i 1.0 Ria antise~e 25.0 r 8.8 1.4 22.6 f 8.8 0.9 0.6 RIt~ antismse 24.0 ; 2.6 1,4 26.8 3.9 0.7 i.0 REQ atd Rti~ antisense 19.0 ~ 5.9 1.1 tp.t + 8.2 1.0 0.5 Cells were.exposed to each of 15 ~uM concentrations of RIB, RII~r or RIa and RII~ antisense oligodeoxynucleo-tide for 4 days as shown in fig. 1A. fihe data represent an average ~ SD of duplicate determinations of three identical experiments.
*Picomoles phosphate transferred to ICemptide per min/mg protein.
Example 2 Next, the RIa antisense oligonucleotide having SEQ ID NO:l was administered to mice having an experimental tumor. A pellet of RI« antisense oligonucleotide (25 mgiKg) and cholesterol (1000 mg/Kg) was implanted s.c. in the left flank of athymic mice which had been injected in the right flank with 'LS-174T human colon cancer cells (2x106 cells) suspended in phosphate-buffered='saline. Tumor measurements and mouse weights were recorded on the initial day of treatment 4staging day), and at the end of treatment (staging day +5). the mean tumor weight zg change {D), was based on length and width measurements in millimeters. After a few days, the tumor growth was inhibited when compared to control cells (see Table 3}. No change in body weight was noted in the control and treated animals.
Table 3. Effect of RIa antisense oiigodeoxynucleo-tide s.c, pellet ow the growth of IsS-174T human colon carcinoma in athymic mice intci~t o~~, Fsm ~~,d x cucao~ ut tag) tuno~ wt tm9) at~ac~
Treatments s.c. pellet implanted Control 25 450 --RIa antisense 25 230 48.
(0.5 mg) 8--C1 cAMP {1 mg}b34 250 51 Nsbenzyl cAMP (1 mg) a. 20 mg pellet lyophilized consisting of indicated doses of RIa antisense or cAMP analogs plus supplement doses of cholesterol.
b. The growth inhibitory effect of these cAMP
analogs correlate with decrease in RIa (Natl_._, Cancer Inst. 81 982 {198_9}) and is shown here for comparison.
c. Mean tumor weight per group (4 mice) on staging_day.
d. Mean tumor weight per group on staging day +5.
e. % of change in test tumor weight (aT)/change in control tumor weight (~C).
In other in vitro experiments, the RI« antisense oligonucleotide having SEQ ID NO: 1 was added to dishes containing neuroblastoma,=''colon carcinonea, breast carcinoma and gastric carcinoma cells. A;:
shown in Figure 5, the RIa antisense oligonucleotide having SEQ ID No: 1, inhibited proliferation of uj' cancer cell types When compared to control cells.
Moreover, the Rra antisense oligonucleotide having SEQ
. ID No: 1 caused differentiatioai of the human neuroblastoma cells (see Figure 6).
Examaple 3 -Next, the effect of O-oligo and S-oligo RIB
antisanse oligonucleotides an the growth of LS-174T
human colon carcinoma in athymic mice was compared.
Materials and Methods. We synthesized_[Milligen Biosearch 8700 DNA synthesizer (Bedford, MA)] the 21-mer antisense oligodeoxynucleotides and their phosphorothioate analogs complementary to the human RIa, human RII~ mRIIA transcripts starting from the first colon, and mismatched sequence (random) oligomers of identical sire. The aligomers had the following sequences: RIa antisense, 5~-GGC-GG~°-ACT-GCC-AGA-CTC-CAT-3 ~ (SEQ ID No: I); Fttl~ antisense, i'-CGC-CGG-GAT-CTC-GAT-GCT-CAT-3' (SEQ ID No: 8~i and random oligo, 5'-CGA-TCG-ATC-GAT-CGA-TCG-TAC-3 ~ (SEQ ID No: 7).
LS-174T human colon carcinoma cells (2 x 106) Were injected s.c. in athymic mice, and the antisense oligodeoxynucleotides in the form of either a cholesterol pellet or 50% sesame oil emulsion were administered s.c_ 1 week later when mean tumor sizes usually were 25-50 mg. Tumor volume was based on.
length and width measurements and calculated by the formula 4/3 rte, where r ~ (length~+ width)/4.
Results and Disaussioa: Fig. 7 shows the dose-and time-dependent affect of an RIa antisense oligodeoxynucleotide (o--oligo) at 0.2 and 0.5 mq doses in cholesterol pellets administered s.c. one time (at zero time); it' brought about. 20 and 46% growth inhibition, respectively, in 7 days, When compared with control (untreated) tumors (Fig. 7A). Strikingly, f~he RIa antisense phosphorothioate analog (S-oligo) at a 0.2 mg dose (cholesterol pelT.et, s.c.) gave a 60%
growth inhibition at day 7, exhibiting a 3-fold greater potency than the 0-oliga antisense (Fig. 7A).
The growth inhibitory effect of RIa antisense S-oligo was even greater when animals were treated for a longer period: The Rla antisense S-oligo at a 0.3 mg dose in a cholesterol pellet, ~2 timesJweek s.c.
implantation for 3 weeks, resulted in a 80% growth inhibition; the tumor growth almost stopped after 2 weeks of treatment (Fig. ?B). RIa antisense O-oligo ZO or S-oligo administered s.c. as 50% sesame oil emulsion gave similar results. RIa antisense S-oligo brought ahout no apparent toxicity in animals; no body weight loss or other toxic symptoms were observed during the 3 weeks of treatment.
' The growth inhibitory effect brought about_by RIa antisense S-oligo was the specific effect of the oligomer: RII~ antisense or random (mismatched sequence) S-oligos of the identical size as the RIa antisense oligomer had no effect on the tumor growth (Fig. ?B).
To provide more evidence that the growth inhibition observed in colon carcinomas in athymic mice treated with RIa antisense oligodeoxynucleotide was due to an intracellular effect of the oligomer, the levels of Rh and RII~ cAMP receptor proteins in these tumors were determined. RIa levels were determined~by immunoblotting {Ally, S., Proc. Hall.
cad. Sci. USA 85:6319-5322 (1988)) using monoclonal antibody against human Rla (kindly provided by Drs.
T. I~ea, University of Oslo, Oslo, Norway, and S.O. Deskeland, University of Bergen, Bergen, Norway), and RII~ was measured by immunoprecipitation (Tortora;
G., et al., Proc. Natl. Aced. Sci. USA 87:705-?08 (1990)) with anti-RIT~ antiserum (kindly provided by Dr. S.t?. Deskeland) after ghotoaffinity lak~eling of RII~ with [32P]. 8-H3-cAMP. As shown in Table 4, RIa antisense s-oligomer treatment brought about a marked - zs -reduction (80% decrease) of RIB level in tumors as compared with that in untreated control tumors. This suppression of RIa expression by Rx« antisense S-oligomer brought about a 2-fold increase in RII~ level (Table 4 ) . Such coordinated expression of R:Ia and RThg without changes in the amount of catalytic subunit of protein kinase has been shown in HI.-60 leukemia cells that demonstrated growth inhibition and differentiation upon exposure to RIa antisense oligodeoxynucleotide. On the other hand, a 50%
increase in RIa level along with 80% suppression in RII~ level was observed in tumors after treatment with RII~g antisense S-oligomer (Table 4) which had no effect on tumor growth (Fig. 7). Random (mismatched 3.5 secquencej S-oligomer which had no effect on, tumor growth (Fig. 7) also showed no effect on RIa levels (Table 4j. Thus, reduction in RIa expression appears to trigger a decrease or halt in tumor growth upon treatment with RIa antisense oligomer. Our results demonstrated that cAMP transducer signals fox dual control, either positive or negative, on cell proliferation, depending on the availability of RI« or RII~ receptor proteins. The RIQ antisense oligodeoxynucleotide, which suppressed RIa and enhanced RII~ expression, led to inhibition of in vivo growth of solid colon carcinoma in athymic mice with no symptoms of toxicity in animals. The phosphorothioate analog (S-oligo). of RI,~ antisense oligomer exhibited a greater potency than the antisense of unmodified oligodeoxynucleotide (O-oligo). It has been shown that S-oligos, as compared with O-oligos, more readily enter cells, are more resistant to e:idonucleases, and yet exhibit high efficacy in hybridization with target mRNAs or DNAc (Stein, C.A., et al., In: J.S. Cohen (ed.jr Olirxodeoxynucleotides: ~ Antiser~se Inhibitors of Gene _ 29 Expression, pp. 97-117. Boca Raton, FL, CRC Press, Inc. (1989)).
These results demonstrate here for the first time the striking in vivo effect of antisense oligodeoxynucleotide in the suppression of, malignancy.
The depletion of RId, the type I regulatory subunit of cAMP-dependent protein kinase, by means of an antisense oligodeoxynucleotide, especially with its phosphorothioate analog,.leads to a successful halt of 30 tumor growth in vivo with no symptoms of toxicity, suggesting great potential of this antisense oligodeoxynucleotide fox clinical application.
Table 4 Suppression of RIa cAMP Receptor Expression by RI«
Antisense Oligodeoxynucleotide (s-oligo) Results in Compensatory Increase in RTIa Receptor Relative Levels Treatment RIB RII~
None 1.0 -1 0.1 1.0 ~ 0.1 RI« antisense S-oligo 0.2 -1 0.03 2.0 ~ 0.2 RIIB antisense S-oligo 1.5 ~ 0.2 0.2 ~ 0.02 Random S-~oligo 1.0 -i 0.1 1.0 ~ 0.1 Treatment with S-oligos as indicated were the same as that in Fig. 7B. At the end of the experiment (3 ~0 weeks), tumor extracts were prepared as previously described (Ally, S. et al., Cancer Res. 49:5650-5655 .(1980)) and immunoblotting and immunoprecipitation of RTa ,and RII~, respectively, were performed as previously described by Ally, S., et al., Proc. Natl.
Acad. Sci. USA 85:6319-6322 (1988) and Tortora, G., et al., Proc_ Natl. Ac~,d. Sci. USA 87:705-708_ (2990).
Data are from quantification by densitometric scanning of autoradiograms. Data are expressed relative to levels in control tumors (no treatment), which are set to equal to one as an arbitrary unit.
Data represent an average ~ S.D. of 7 tumors.
_ ~a In the following sequence listing, Seq ID No: 1 represents an antisense sequence corresponding to the first 7 N-terminal colons for RIa. Seq ID No: 2 represents an.antisense sequence corresponding to the 8th-13th colon for RI«. Seq ID No: 3~ represents an antisense sequence corresponding to the 14th-20th colon for RIa. Seq ID No: 4 represents an antisense ..~~ _ sequence corresponding to the 94th-100th colon for RIB.
Seq ID No: 5 represents an antisense sequence corresponding to the lgt-100th colon for RIa. Seq ID
No: 6 represents the sense sequence corresponding to the 18t-100th colon for RI«.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Yoon S. Cho-Chung (ii) TITLE OF INVENTION: Antisense Oligonucleotides for Treatment of Cancer (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MBM & CO.
(B) STREET: P.O. BOX 809, STATION B
(C) CITY: OTTAWA
(D) PROVINCE: ONTARIO
(E) COUNTRY: CANADA
(F) POSTAL CODE: K1P 5P9 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible {C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Ver. 2.1 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,054,325 (B) FILING DATE: 1991-10-28 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 607,113 (B) FILING DATE: 1990-11-02 {C) CLASSIFICATION:
(A) APPLICATION NUMBER: US 680,198 (B) FILING DATE: 1991-04-05 (C) CLASSIFICATION:
(A) APPLICATION NUMBER: US 702,163 (B) FILING DATE: 1991-05-20 {C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SWAIN, Margaret (B) REGISTRATION NUMBER: 10926 (C) REFERENCE/DOCKET NUMBER: 127-115 {ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 613/567-0762 (B) TELEFAX: 613/563-7671 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense DNA complementary to Protein Kinase subunit RIa mRNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
INFORMATTON FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: No (vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo Sapiens (ix) FEATURE:
{A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Sequence: Partial sequence of Protein Kinase Regulatory Subunit RIa (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleio Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(iv) ANTISENSE: Yes (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: Description of Artificial Sequence: Antisense oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Claims (30)
1. A pharmaceutical composition comprising antisense oligonucleotide having the nucleotide sequence as set forth in SEQ ID NO:1 and a pharmaceutically acceptable carrier.
2. The pharmaceutical composition of claim 1, wherein said antisense oligonucleotide is DNA, RNA or a combination thereof.
3. The pharmaceutical composition of claim 1 or 2, wherein said antisense oligonucleotide is a 5' to 3'-oligodeoxynucleotide.
4. The pharmaceutical composition of any one of claims 1 - 3, wherein said antisense oligonucleotide comprises internucleotide linkages that are selected from the group of phosphodiesters or phosphorothioate phosphodiesters.
5. The pharmaceutical composition of any one of claims 1 - 4, wherein said pharmaceutically acceptable carrier is a sterol.
6. The pharmaceutical composition of any one of claims 1 - 4, wherein said pharmaceutically acceptable carrier is a liposome.
7. Use of antisense oligonucleotide having the nucleotide sequence as set forth in SEQ ID NO:1 in the preparation of a pharmaceutical composition for the treatment of cancers.
8. The use according to claim 7, wherein said cancer is selected from the group of gastric, pancreatic, lung, breast, anal, colorectal, head and neck neoplasms, neuroblastomas melanoma, sarcoma and carcinoma.
9. The use according to claim 7, wherein said cancer is selected from the group of F9 teratocarcinoma, SK-N-SH
neuroblastoma, TMK-1 gastric carcinoma, Mecca lymphosarcoma, Gardner lymphosarcoma, Ridgway Osteogenic sarcoma, Sarcoma 180 (ascites), Wagner osteogenic sarcoma, Sarcoma T241, Lewis lung carcinoma, Carcinoma 755, CD8F, MCF-7 breast carcinoma, Colon 38, LS-174T
colon carcinoma, Carcinoma 1025, Erhlich carcinoma (ascites & solid), Krubs 2 carcinoma (ascites), Bashford carcinoma 63, Adenocarcinoma E 0771, B16 Melanoma, Hardin-Passey melanoma, Giloma 26, Miyona adenocarcinoma, Walker carcinosarcoma 256, Flexner-Jobling carcinoma, Jensen sarcoma, Iglesias sarcoma, Iglesias ovarian tumor, Murphy-Sturn lymphosarcoma, Yoshida sarcoma, Dunning leukemia, Rous chicken sarcoma, and Crabb hamster sarcoma.
neuroblastoma, TMK-1 gastric carcinoma, Mecca lymphosarcoma, Gardner lymphosarcoma, Ridgway Osteogenic sarcoma, Sarcoma 180 (ascites), Wagner osteogenic sarcoma, Sarcoma T241, Lewis lung carcinoma, Carcinoma 755, CD8F, MCF-7 breast carcinoma, Colon 38, LS-174T
colon carcinoma, Carcinoma 1025, Erhlich carcinoma (ascites & solid), Krubs 2 carcinoma (ascites), Bashford carcinoma 63, Adenocarcinoma E 0771, B16 Melanoma, Hardin-Passey melanoma, Giloma 26, Miyona adenocarcinoma, Walker carcinosarcoma 256, Flexner-Jobling carcinoma, Jensen sarcoma, Iglesias sarcoma, Iglesias ovarian tumor, Murphy-Sturn lymphosarcoma, Yoshida sarcoma, Dunning leukemia, Rous chicken sarcoma, and Crabb hamster sarcoma.
10. The use according to any one of claims 7 - 9, wherein said antisense oligonucleotide is DNA, RNA or a combination thereof.
11. The use according to any one of claims 7 - 9, wherein said antisense oligonucleotide is a 5' to 3'-oligodeoxynucleotide.
12. The use according to any one of claims 7 - 11, wherein said antisense oligonucleotide has internucleotide linkages that are selected from the group of phosphodiesters or phosphorothioate phosphodiesters.
13. The use according to any one of claims 7 - 12, wherein said pharmaceutical composition comprises a pharmaceutically acceptable carrier.
14. The use according to claim 13, wherein said pharmaceutically acceptable carrier is a sterol.
15. The use according to claim 13, wherein said pharmaceutically acceptable carrier is a liposome.
16. The use according to any one of claims 7 - 15, wherein said antisense oligonucleotide is combined with an agent which enhances uptake of said antisense oligonucleotide by cells.
17. The use according to claim 16, wherein the agent is a selected from the group of a lipophilic cationic compound, a lipophilic carrier or a peptide, that is ingested by cells.
18. Use of a therapeutically effective amount of antisense oligonucleotide having the nucleotide sequence as set forth in SEQ ID NO:1 in the treatment of cancer.
19. The use according to claim 18, wherein the cancer is selected from the group of gastric, pancreatic, lung, breast, anal, colorectal, head and neck neoplasms, neuroblastomas, melanoma, sarcoma and carcinoma.
20. The use according to claim 18, wherein said cancer is selected from the group of F9 teratocarcinoma, SK-N-SH
neuroblastoma, TMK-1 gastric carcinoma, Mecca lymphosarcoma, Gardner lymphosarcoma, Ridgway Osteogenic sarcoma, Sarcoma 180 (ascites), Wagner osteogenic sarcoma, Sarcoma T241, Lewis lung carcinoma, Carcinoma 755, CDBF, MCF-7 breast carcinoma, Colon 38, LS-174T
colon carcinoma, Carcinoma 1025, Erhlich carcinoma (ascites & solid), Krubs 2 carcinoma (ascites), Bashford carcinoma 63, Adenocarcinoma E 0771, B16 Melanoma, Hardin-Passey melanoma, Giloma 26, Miyona adenocarcinoma, Walker carcinosarcoma 256, Flexner-Jobling carcinoma, Jensen sarcoma, Iglesias sarcoma, Iglesias ovarian tumor, Murphy-Sturn lymphosarcoma, Yoshida sarcoma, Dunning leukemia, Rous chicken sarcoma, and Crabb hamster sarcoma.
neuroblastoma, TMK-1 gastric carcinoma, Mecca lymphosarcoma, Gardner lymphosarcoma, Ridgway Osteogenic sarcoma, Sarcoma 180 (ascites), Wagner osteogenic sarcoma, Sarcoma T241, Lewis lung carcinoma, Carcinoma 755, CDBF, MCF-7 breast carcinoma, Colon 38, LS-174T
colon carcinoma, Carcinoma 1025, Erhlich carcinoma (ascites & solid), Krubs 2 carcinoma (ascites), Bashford carcinoma 63, Adenocarcinoma E 0771, B16 Melanoma, Hardin-Passey melanoma, Giloma 26, Miyona adenocarcinoma, Walker carcinosarcoma 256, Flexner-Jobling carcinoma, Jensen sarcoma, Iglesias sarcoma, Iglesias ovarian tumor, Murphy-Sturn lymphosarcoma, Yoshida sarcoma, Dunning leukemia, Rous chicken sarcoma, and Crabb hamster sarcoma.
21. The use according to any one of claims 18 - 20, wherein said antisense oligonucleotide, or derivatives thereof, are DNA, RNA or a combination thereof.
22. The use according to any one of claims 18 - 21, wherein said antisense oligonucleotide is 5' to 3'-oligodeoxynucleotide.
23. The use according to any one of claims 18 - 22, wherein said antisense oligonucleotide has internucleotide linkages that are selected from the group of phosphodiester or phosphorothioate phosphodiesters.
24. The use according to any one of claims 18 - 23, wherein said antisense oligonucleotide is contained in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
25. The use according to claim 24, wherein said pharmaceutically acceptable carrier is a sterol.
26. The use according to claim 24, wherein said pharmaceutically acceptable carrier is a liposome.
27. The use according to any one of claims 18 - 26, wherein said antisense oligonucleotide is combined with an agent which enhances uptake of said antisense oligonucleotide by cells.
28. The use according to claim 27; wherein the agent is a selected from the group of a lipophilic cationic compound, a lipophilic carrier or a peptide, that is ingested by cells.
29. Use of antisense oligonucleotide having the nucleotide sequence as set forth in SEQ ID NO:1, for the preparation of a pharmaceutical composition for the treatment of a cancer selected from the group consisting of gastric, pancreatic, lung, breast, anal, colorectal, head and neck neoplasms, neuroblastomas and melanoma.
30. Use of antisense oligonucleotide having the nucleotide sequence as set forth in SEQ ID NO:1, for administration to a patient having cancer selected from the group consisting of gastric, pancreatic, lung, breast, anal, colorectal, head and neck neoplasms, neuroblastomas and melanoma.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US60711390A | 1990-11-02 | 1990-11-02 | |
| US607,113 | 1990-11-02 | ||
| US68019891A | 1991-04-05 | 1991-04-05 | |
| US680,198 | 1991-04-05 | ||
| US07/702,163 US5271941A (en) | 1990-11-02 | 1991-05-20 | Antisense oligonucleotides of human regulatory subunit RI.sub.α of cAMP-dependent protein kinases |
| US702,163 | 1991-05-20 | ||
| CA002054325A CA2054325C (en) | 1990-11-02 | 1991-10-28 | Antisense oligonucleotides for treatment of cancer |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002054325A Division CA2054325C (en) | 1990-11-02 | 1991-10-28 | Antisense oligonucleotides for treatment of cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2488792A1 true CA2488792A1 (en) | 1992-05-03 |
Family
ID=27416969
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002488792A Abandoned CA2488792A1 (en) | 1990-11-02 | 1991-10-28 | Antisense oligonucleotides for treatment of cancer |
| CA002054325A Expired - Fee Related CA2054325C (en) | 1990-11-02 | 1991-10-28 | Antisense oligonucleotides for treatment of cancer |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002054325A Expired - Fee Related CA2054325C (en) | 1990-11-02 | 1991-10-28 | Antisense oligonucleotides for treatment of cancer |
Country Status (9)
| Country | Link |
|---|---|
| US (3) | US5271941A (en) |
| EP (3) | EP0490077B1 (en) |
| KR (1) | KR0171210B1 (en) |
| AT (2) | ATE194383T1 (en) |
| CA (2) | CA2488792A1 (en) |
| DE (2) | DE69127175T2 (en) |
| DK (2) | DK0785252T3 (en) |
| ES (2) | ES2148838T3 (en) |
| GR (2) | GR3024673T3 (en) |
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| JP7237064B2 (en) | 2017-09-06 | 2023-03-10 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | Single immunoglobulin interleukin-1 receptor-related (SIGIRR) variants and uses thereof |
| CA3074682A1 (en) | 2017-09-07 | 2019-03-14 | Regeneron Pharmaceuticals, Inc. | Solute carrier family 14 member 1 (slc14a1) variants and uses thereof |
| AU2018348195A1 (en) | 2017-10-11 | 2020-04-23 | Regeneron Pharmaceuticals, Inc. | Inhibition of HSD17b13 in the treatment of liver disease in patients expressing the PNPLA3 I148m variation |
| EP3697807A1 (en) | 2017-10-16 | 2020-08-26 | Regeneron Pharmaceuticals, Inc. | Cornulin (crnn) variants and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5190931A (en) * | 1983-10-20 | 1993-03-02 | The Research Foundation Of State University Of New York | Regulation of gene expression by employing translational inhibition of MRNA utilizing interfering complementary MRNA |
| NO864301L (en) * | 1986-10-28 | 1988-04-29 | Tore Jahnsen | DNA PROBLEMS FOR HUMAN REGULATORY UNIT OF CAMP-DEPENDED PROTEIN KINASES. |
| WO1990000624A1 (en) * | 1988-07-05 | 1990-01-25 | Baylor College Of Medecine | Antisense oligonucleotide antibiotics complementary to the macromolecular synthesis operon, methods of treating bacterial infections and methods for identification of bacteria |
| US5098890A (en) * | 1988-11-07 | 1992-03-24 | Temple University-Of The Commonwealth System Of Higher Education | Antisence oligonucleotides to c-myb proto-oncogene and uses thereof |
| US5087617A (en) * | 1989-02-15 | 1992-02-11 | Board Of Regents, The University Of Texas System | Methods and compositions for treatment of cancer using oligonucleotides |
| US5271941A (en) * | 1990-11-02 | 1993-12-21 | Cho Chung Yoon S | Antisense oligonucleotides of human regulatory subunit RI.sub.α of cAMP-dependent protein kinases |
-
1991
- 1991-05-20 US US07/702,163 patent/US5271941A/en not_active Expired - Lifetime
- 1991-10-28 CA CA002488792A patent/CA2488792A1/en not_active Abandoned
- 1991-10-28 CA CA002054325A patent/CA2054325C/en not_active Expired - Fee Related
- 1991-10-31 AT AT97100277T patent/ATE194383T1/en not_active IP Right Cessation
- 1991-10-31 AT AT91118628T patent/ATE156517T1/en not_active IP Right Cessation
- 1991-10-31 ES ES97100277T patent/ES2148838T3/en not_active Expired - Lifetime
- 1991-10-31 DK DK97100277T patent/DK0785252T3/en active
- 1991-10-31 ES ES91118628T patent/ES2104644T3/en not_active Expired - Lifetime
- 1991-10-31 DE DE69127175T patent/DE69127175T2/en not_active Expired - Fee Related
- 1991-10-31 EP EP91118628A patent/EP0490077B1/en not_active Expired - Lifetime
- 1991-10-31 EP EP97100277A patent/EP0785252B1/en not_active Expired - Lifetime
- 1991-10-31 EP EP99119780A patent/EP0972831A1/en not_active Withdrawn
- 1991-10-31 DK DK91118628.6T patent/DK0490077T3/en active
- 1991-10-31 DE DE69132299T patent/DE69132299T2/en not_active Expired - Fee Related
- 1991-11-02 KR KR1019910019468A patent/KR0171210B1/en not_active IP Right Cessation
-
1993
- 1993-05-14 US US08/060,984 patent/US5627158A/en not_active Expired - Fee Related
-
1995
- 1995-02-02 US US08/383,742 patent/US5691317A/en not_active Expired - Fee Related
-
1997
- 1997-09-10 GR GR970402319T patent/GR3024673T3/en unknown
-
2000
- 2000-10-02 GR GR20000402225T patent/GR3034535T3/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| DE69132299T2 (en) | 2001-02-15 |
| GR3034535T3 (en) | 2001-01-31 |
| ATE194383T1 (en) | 2000-07-15 |
| DK0490077T3 (en) | 1998-01-26 |
| EP0490077A1 (en) | 1992-06-17 |
| US5691317A (en) | 1997-11-25 |
| EP0490077B1 (en) | 1997-08-06 |
| CA2054325A1 (en) | 1992-05-03 |
| ATE156517T1 (en) | 1997-08-15 |
| US5627158A (en) | 1997-05-06 |
| DK0785252T3 (en) | 2000-10-02 |
| DE69127175T2 (en) | 1998-03-12 |
| ES2104644T3 (en) | 1997-10-16 |
| EP0785252A1 (en) | 1997-07-23 |
| CA2054325C (en) | 2005-03-15 |
| US5271941A (en) | 1993-12-21 |
| EP0972831A1 (en) | 2000-01-19 |
| DE69132299D1 (en) | 2000-08-10 |
| GR3024673T3 (en) | 1997-12-31 |
| KR0171210B1 (en) | 1999-02-01 |
| EP0785252B1 (en) | 2000-07-05 |
| DE69127175D1 (en) | 1997-09-11 |
| ES2148838T3 (en) | 2000-10-16 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued |