U.S. patent application number 11/586993 was filed with the patent office on 2007-02-15 for oligonucleotide n3'-p5' thiophosphoramidates: their synthesis and use.
Invention is credited to Sergei Gryaznov, Tracy Matray, Krisztina Pongracz.
Application Number | 20070037770 11/586993 |
Document ID | / |
Family ID | 26850281 |
Filed Date | 2007-02-15 |
United States Patent
Application |
20070037770 |
Kind Code |
A1 |
Gryaznov; Sergei ; et
al. |
February 15, 2007 |
Oligonucleotide N3'-P5' thiophosphoramidates: their synthesis and
use
Abstract
Oligonucleotides with a novel sugar-phosphate backbone
containing at least one internucleoside 3'-NHP(O)(S.sup.-)O-5'
linkage, and methods of synthesizing and using the inventive
oligonucleotides are provided. The inventive thiophosphoramidate
oligonucleotides were found to retain the high RNA binding affinity
of the parent oligonucleotide N3'.fwdarw.P5' phosphoramidates and
to exhibit a much higher acid stability.
Inventors: |
Gryaznov; Sergei; (San
Mateo, CA) ; Pongracz; Krisztina; (Oakland, CA)
; Matray; Tracy; (San Lorenzo, CA) |
Correspondence
Address: |
GERON CORPORATION
230 CONSTITUTION DRIVE
MENLO PARK
CA
94025
US
|
Family ID: |
26850281 |
Appl. No.: |
11/586993 |
Filed: |
October 25, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10967755 |
Oct 18, 2004 |
7138383 |
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11586993 |
Oct 25, 2006 |
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10463076 |
Jun 17, 2003 |
6835826 |
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10967755 |
Oct 18, 2004 |
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09657445 |
Sep 8, 2000 |
6608036 |
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10463076 |
Jun 17, 2003 |
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60153201 |
Sep 10, 1999 |
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60160444 |
Oct 19, 1999 |
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Current U.S.
Class: |
514/44A ;
435/6.12; 536/23.1 |
Current CPC
Class: |
C12Q 1/6813 20130101;
C12Y 207/07049 20130101; C12N 2320/10 20130101; C07H 1/06 20130101;
C12N 2310/314 20130101; C12Q 1/6832 20130101; C12N 2320/50
20130101; C12N 2320/51 20130101; A61P 43/00 20180101; C07H 21/00
20130101; C12N 15/1137 20130101; C12N 15/111 20130101; C12N 15/113
20130101; C12N 2330/30 20130101; A61P 31/12 20180101; A61P 35/00
20180101 |
Class at
Publication: |
514/044 ;
435/006; 536/023.1 |
International
Class: |
A61K 48/00 20070101
A61K048/00; C12Q 1/68 20060101 C12Q001/68; C07H 21/02 20060101
C07H021/02 |
Claims
1. A polynucleotide comprising a non-homopolymeric sequence of
nucleoside subunits joined by at least one inter-subunit linkage
that is a N3'.fwdarw.P5' thiophosphoramidate.
2. The polynucleotide of claim 1, wherein the N3'.fwdarw.P5'
thiophosphoramidate linkages is defined by the formula:
3'-[--NH--P(.dbd.O)(--SR)--O--]-5': wherein R is selected from the
group consisting of hydrogen, alkyl, aryl and salts thereof.
3. The polynucleotide of claim 2, wherein all intersubunit linkages
are N3'.fwdarw.P5' thiophosphoramidate linkages.
4. The polynucleotide of claim 3, wherein the defined sequence of
the nucleoside subunits has a length of from 3 to 50.
5. The polynucleotide of claim 1, comprising a second linkage
selected from the group consisting of phosphodiester,
phosphotriester, methylphosphonate, P3'.fwdarw.N5' phosphoramidate,
N3'.fwdarw.P5' phosphoramidate, and phosphorothioate.
6. The polynucleotide of claim 5, which is 3 to 50 nucleotide
subunits in length.
7. A polynucleotide comprising a sequence of nucleoside subunits
containing at least one subunit defined by the formula: ##STR12##
wherein B is a purine or pyrimidine or an analog thereof; Z is OR,
SR, or methyl, wherein R is selected from the group consisting of
hydrogen, alkyl, aryl and salts thereof; and R.sub.1 is selected
from the group consisting of hydrogen, O--R.sub.2, S--R.sub.2, and
halogen, wherein R.sub.2 is H, alkyl, or
(CH.sub.2).sub.nW(CH.sub.2).sub.mH, where n is between 1-10, m is
between 0-10 and W is O, S, or NH, with the proviso that when Z is
methy or OMe, R.sub.1 is not H.
8. The polynucleotide of claim 7, wherein all subunits are defined
by the formula: ##STR13##
9. The polynucleotide of claim 7, wherein the polynucleotide
further comprises at least one subunit selected from the group
consisting of phosphodiester, phosphotriester, methylphosphonate,
P3'.fwdarw.N5' phosphoramidate, N3'.fwdarw.P5' phosphoramidate, and
phosphorothioate subunits.
10. The polynucleotide of claim 7, wherein each B moiety in the
polynucleotide is independently selected from the group consisting
of uracil, thymine, adenine, guanine, cytosine, 5-methylcytosine,
5-bromouracil, and inosine.
11. The polynucleotide of claim 7, wherein the polynucleotide
hybridizes with a target nucleic acid sequence.
12. The polynucleotide of claim 11, wherein the target nucleic acid
sequence is a sequence of telomerase RNA component.
13. The polynucleotide of claim 10, wherein the polynucleotide can
hybridize with an RNA target.
14. The polynucleotide of claim 7, wherein the polynucleotide
comprises a reporter moiety.
15. The polynucleotide of claim 14, wherein the reporter moiety is
selected from the group consisting of radioactive labels, biotin
labels, and fluorescent labels.
16. A method of synthesizing an oligonucleotide N3'.fwdarw.P5'
thiophosphoramidate, the method comprising: (a) providing a first
5'-succinyl-3'-aminotrityl-2',3'-dideoxy nucleoside attached to a
solid phase support, the first nucleoside having a protected 3'
amino group; (b) deprotecting the protected 3' amino group to form
a free 3' amino group; (c) reacting the free 3' amino group with a
3'-protected
aminonucleoside-5'-O-cyanoethyl-N,N-diisopropylaminophosphoramidite
monomer to form an internucleoside N3'.fwdarw.P5' phosphoramidite
linkage; and (d) sulfurizing the internucleaside phosphoramidite
group to form a N3'.fwdarw.P5' thiophosphoramidate.
17. A method according to claim 16, wherein reacting and
sulferizing are repeated.
18. A method of hybridizing polynucleotide to a DNA or RNA target
comprising contacting a polynucleotide according to claim 1 with
the target under conditions that allow formation of a hybridization
complex between the polynucleotide and the target.
19. A method according to claim 18, wherein the polynucleotide
carries a reporter moiety.
20. A method according to claim 19, wherein the reporter moiety is
selected from the group consisting of radioactive labels, biotin
labels, and fluorescent labels.
21. A method for determining a nucleic acid containing a specific
sequence in a sample, comprising: a) preparing a reaction mixture
comprising the sample and a polynucleotide according to claim 1
capable of hybridizing specifically with the sequence; b)
determining hybrids formed in the reaction mixture; and c)
correlating any hybrids formed with nucleic acid containing the
specific sequence in the sample.
22. A method for isolating a nucleic acid containing a specific
sequence from a sample, comprising: a) combining the sample and a
polynucleotide according to claim 1 capable of hybridizing
specifically with the sequence; and b) recovering the nucleic acid
from hybrids formed with the polynucleotide.
23. A method for inhibiting function of an RNA in a cell,
comprising contacting the cell with a polynucleotide according to
claim 1 that can specifically hybridize with the RNA.
24. A method according to claim 23, which is a method for
inhibiting translation of an mRNA, wherein the polynucleotide
comprises a sequence containing at least 10 bases complementary to
a sequence contained in the mRNA.
25. A method according to claim 23, which is a method for
inhibiting telomerase enzyme in a cell, wherein the polynucleotide
comprises a sequence complementary to telomerase RNA component.
26. A method for inhibiting activity of a telomerase enzyme in a
cell comprising contacting the cell with an effective amount of a
polynucleotide according to claim 1.
27. A kit for determining or isolating a nucleic acid containing a
specific sequence in a sample, comprising a polynucleotide
according to claim 1 that can hybridize to the specific sequence,
and written indications for using the polynucleotide for
determining or isolating the nucleic acid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/967,755, filed Oct. 18, 2004, allowed, which is a continuation
of U.S. Ser. No. 10/463,076, filed Jun. 17, 2003, pending, which is
a continuation of U.S. Ser. No. 09/657,445, filed Sep. 8, 2000, now
U.S. Pat. No. 6,608,036; which claimed priority from U.S. Ser. No.
60/153,201, filed Sep. 10, 1999 and U.S. Ser. No. 60/160,444, filed
Oct. 19, 1999. All of the above-referenced applications are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to oligonucleotides having a
novel sugar-phosphate backbone containing internucleoside
3'-NHP(O)(S.sup.-)O-5' linkages. More particularly, the present
invention is directed to thiophosphoramidate oligonucleotide
compositions, their use as diagnostic or therapeutic agents and
methods for synthesizing thiophosphoramidate oligonucleotides.
BACKGROUND OF THE INVENTION
[0003] Nucleic acid polymer chemistry has played a crucial role in
many developing technologies in the pharmaceutical, diagnostic, and
analytical fields, and more particularly in the subfields of
antisense and anti-gene therapeutics, combinatorial chemistry,
branched DNA signal amplification, and array-based DNA diagnostics
and analysis (e.g. Uhlmann and Peyman, Chemical Reviews, 90:
543-584, 1990; Milligan et al., J. Med. Chem. 36: 1923-1937, 1993;
DeMesmaeker et al., Current Opinion in Structural Biology, 5:
343-355, 1995; Roush, Science, 276: 1192-1193, 1997; Thuong et al.,
Angew. Chem. Int. Ed. Engl., 32: 666-690, 1993; Brenner et al.,
Proc. Natl. Acad. Sci., 89: 5381-5383, 1992; Gold et al., Ann. Rev.
Biochem., 64: 763-797, 1995; Gallop et al., J. Med. Chem. 37:
1233-1258, 1994; Gordon et al., J. Med. Chem. 37: 1385-1401, 1994;
Gryaznov, International application PCT/US94/07557; Urdea et al.,
U.S. Pat. No. 5,124,246; Southern et al., Genomics, 13: 1008-1017,
1992; McGall et al., U.S. Pat. No. 5,412,087; Fodor et al., U.S.
Pat. No. 5,424,186; Pirrung et al., U.S. Pat. No. 5,405,783).
[0004] Much of this chemistry has been directed to improving the
binding strength, specificity, and nuclease resistance of natural
nucleic acid polymers, such as DNA. Unfortunately, improvements in
one property, such as nuclease resistance, often involve trade-offs
against other properties, such as binding strength. Examples of
such trade-offs abound: peptide nucleic acids (PNAs) display good
nuclease resistance and binding strength, but have reduced cellular
uptake in test cultures (e.g. Hanvey et al., Science,
258:1481-1485, 1992); phosphorothioates display good nuclease
resistance and solubility, but are typically synthesized as
P-chiral mixtures and display several sequence-non-specific
biological effects (e.g. Stein et al., Science, 261:1004-1012,
1993); methylphosphonates display good nuclease resistance and
cellular uptake, but are also typically synthesized as P-chiral
mixtures and have reduced duplex stability (e.g. Mesmaeker et al.
(cited above); and so on.
[0005] Recently, a new class of oligonucleotide analog has been
developed having so-called N3'.fwdarw.P5' phosphoramidate
internucleoside linkages which display favorable binding
properties, nuclease resistance, and solubility (Gryaznov and
Letsinger, Nucleic Acids Research, 20:3403-3409, 1992; Chen et al.,
Nucleic Acids Research, 23:2661-2668, 1995; Gryaznov et al., Proc.
Natl. Acad. Sci., 92:5798-5802, 1995; and Gryaznov et al., J. Am.
Chem. Soc., 116:3143-3144, 1994). Phosphoramidate compounds contain
a 3'-amino group at each of the 2'-deoxyfuranose nucleoside
residues replacing a 3'-oxygen atom. The synthesis and properties
of oligonucleotide N3'.fwdarw.P5' phosphoramidates are also
described in Gryaznov et al. U.S. Pat. Nos. 5,591,607; 5,599,922;
5,726,297; and Hirschbein et al., U.S. Pat. No. 5,824,793.
[0006] The oligonucleotide N3'.fwdarw.P5' phosphoramidates form
unusually stable duplexes with complementary DNA and especially RNA
strands, as well as stable triplexes with DNA duplexes, and they
are also resistant to nucleases (Chen et al., Nucleic Acids
Research, 23:2661-2668, 1995; Gryaznov et al., Proc. Natl. Acad.
Sci., 92:5798-5802 1995). Moreover oligonucleotide N3'.fwdarw.P5'
phosphoramidates are more potent antisense agents than
phosphorothioate derivatives both in vitro and in vivo (Skorski, et
al., Proc. Natl. Acad. Sci., 94:3966-3971, 1997). At the same time
the phosphoramidates apparently have a low affinity to the intra-
and extracellular proteins and increased acid liability relative to
the natural phosphodiester counterparts (Gryaznov et al., Nucleic
Acids Research, 24:1508-1514, 1996). These features of the
oligonucleotide phosphoramidates potentially adversely affect their
pharmacological properties for some applications. In particular,
the acid stability of an oligonucleotide is an important quality
given the desire to use oligonucleotide agents as oral
therapeutics.
[0007] In order to circumvent the above described problems
associated with oligonucleotide analogs, a new class of compounds
was sought that embodies the best characteristics from both
oligonucleotide phosphoramidates and phosphorothioates. The present
invention describes the synthesis, properties and uses of
oligonucleotide N3'.fwdarw.P5' thiophosphoramidates.
SUMMARY OF THE INVENTION
[0008] The compositions and methods of the present invention relate
to polynucleotides having contiguous nucleoside subunits joined by
intersubunit linkages. In the polynucleotides of the present
invention, at least two contiguous subunits are joined by a
N3'.fwdarw.P5' thiophosphoramidate intersubunit linkage defined by
the formula of 3'-[--NH--P(.dbd.O)(--SR)--O--]-5', wherein R is
selected from the group consisting of hydrogen, alkyl, aryl and
salts thereof. In a preferred embodiment of the invention, R is
hydrogen or a salt thereof. The inventive polynucleotides can be
composed such that all of the intersubunit linkages are
N3'.fwdarw.P5' thiophosphoramidate. Alternatively, the
polynucleotides of the invention can contain a second class of
intersubunit linkages such as phosphodiester, phosphotriester,
methylphosphonate, P'3.fwdarw.N5' phosphoramidate, N'3.fwdarw.P5'
phosphoramidate, and phosphorothioate linkages.
[0009] An exemplary N3'.fwdarw.P5' thiophosphoramidate intersubunit
linkage has the formula: ##STR1## where B is a purine or pyrimidine
or an analog thereof, Z is OR, SR, or methyl, wherein R is selected
from the group consisting of hydrogen, alkyl, and aryl and their
salts; and R.sub.1 is selected from the group consisting of
hydrogen, O--R.sub.2, S--R.sub.2, and halogen, wherein R.sub.2 is
H, alkyl, or (CH.sub.2).sub.nW(CH.sub.2).sub.mH, where n is between
1-10, m is between 0-10 and W is O, S, or NH, with the proviso that
when Z is methyl or OMe, R.sub.1 is not H. The nucleoside subunits
making up the polynucleotides can be selected to be in a defined
sequence: such as, a sequence of bases complementary to a
single-strand nucleic acid target sequence or a sequence that will
allow formation of a triplex structure between the polynucleotide
and a target duplex. The nucleoside subunits joined by at least one
N3'.fwdarw.P5' thiophosphoramidate intersubunit linkage, as
described above, have superior resistance to acid hydrolysis, yet
retain the same thermal stability as compared to oligonucleotides
having phosphoramidate intersubunit linkages.
[0010] The present invention also includes a method of synthesizing
an oligonucleotide N3'.fwdarw.P5' thiophosphoramidate. In this
method a first nucleoside 5'-succinyl-3'-aminotrityl-2',3'-dideoxy
nucleoside is attached to a solid phase support. The first
nucleoside additionally has a protected 3' amino group. The
protected 3' amino group is then deprotected to form a free 3'
amino group to which a second nucleoside is added. The free 3'
amino group of the first nucleoside is reacted with a 3'-protected
aminonucleoside-5'-O-cyanoethyl-N,N-diisopropylaminophosphoramidite
monomer to form an internucleoside N3'.fwdarw.P5' phosphoramidite
linkage. The internucleaside phosphoramidite group is then
sulfurized to form a N3'.fwdarw.P5' thiophosphoramidate
internucleaside linkage between the first and second
nucleosides.
[0011] In another embodiment of the invention, a method is provided
for hybridizing a thiophosphoramidate oligonucleotide of the
invention to an DNA or RNA target. The thiophosphoramidate
polynucleotide comprises a sequence of nucleoside subunits joined
by at least one subunit defined by the formula: ##STR2##
[0012] where B is a purine or pyrimidine or an analog thereof, Z is
OR, SR, or methyl, and R.sub.1 is selected from the group
consisting of hydrogen, O--R.sub.2, S--R.sub.2, and halogen,
wherein R.sub.2 is H, alkyl, or (CH.sub.2).sub.nW(CH.sub.2).sub.mH,
where n is between 1-10, m is between 0-10 and W is O, S, or NH,
with the proviso that when Z is methyl or OMe, R.sub.1 is not H.
The thiophosphoramidate polynucleotide is contacted with the RNA
target to allow formation of a hybridization complex between the
polynucleotide and the RNA target.
[0013] The present invention also includes pharmaceutical
compositions and kits including a polynucleotide having at least
one N3'.fwdarw.P5' thiophosphoramidate linkage, as described above.
The inventive oligonucleotides are particularly useful in oral
therapeutic applications based on hybridization, such as, antigene
and antisense applications, including the inhibition of telomerase
enzyme activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The foregoing aspects and many of the attendant advantages
of this invention will become more readily appreciated as the same
become better understood by reference to the following detailed
description, when taken in conjunction with the accompanying
drawings, wherein:
[0015] FIG. 1A shows the internucleoside linkage structure of
oligonucleotide phosphorothioates;
[0016] FIG. 1B shows the internucleoside linkage structure of
oligonucleotide phosphoramidates;
[0017] FIG. 1C shows the internucleoside linkage structure of
exemplary oligonucleotide thiophosphoramidates of the
invention.
[0018] FIG. 2 shows a schematic outline of the step-by-step
synthesis of uniformly modified oligonucleotide
thiophosphoramidates.
[0019] FIG. 3 shows a schematic outline of the conversion a
dinucleotide thiophosphoramidate into its phosphoramidate
counterpart, as well as the products resulting from the hydrolysis
of the dinucleotide thiophosphoramidate.
[0020] FIG. 4 shows the results of an in vitro telomerase
inhibition assay performed using increasing amount of
thiophosphoramidate oligonucleotide of SEQ ID NO:2 that is
complementary to telomerase RNA, or SEQ ID NO: 4 that contains
nucleotide mismatches.
[0021] FIG. 5 shows the results of an in vitro telomerase
inhibition assay performed using increasing amount of
thiophosphoramidate oligonucleotide of SEQ ID NO:10 that is
complementary to telomerase RNA.
[0022] FIG. 6 shows the results of SEQ ID NOs: 2 and 10 that are
complementary to telomerase RNA, and SEQ ID NO:4 that contains
nucleotide mismatches on the growth of HME50-5E cells.
[0023] FIG. 7 shows the results of the thiophosphoramidate
oligonucleotides on the telomere length of HME50-5E cells.
[0024] FIG. 8 illustrates the IC.sub.50 values measured for the
thiophosphoramidate oligonucleotide SEQ ID NO: 2.
DETAILED DESCRIPTION
Definitions
[0025] An "alkyl group" refers to an alkyl or substituted alkyl
group having 1 to 20 carbon atoms, such as methyl, ethyl, propyl,
and the like. Lower alkyl typically refers to C.sub.1 to C.sub.5.
Intermediate alkyl typically refers to C.sub.6 to C.sub.10.
[0026] An "aryl group" refers to an aromatic ring group having 5-20
carbon atoms, such as phenyl, naphthyl, anthryl, or substituted
aryl groups, such as, alkyl- or aryl-substitutions like tolyl,
ethylphenyl, biphenylyl, etc. Also included are heterocyclic
aromatic ring groups having one or more nitrogen, oxygen, or sulfur
atoms in the ring.
[0027] "Oligonucleotides" typically refer to nucleoside subunit
polymers having between about 3 and about 50 contiguous subunits.
The nucleoside subunits can be joined by a variety of intersubunit
linkages, including, but not limited to, those shown in FIGS. 1A to
1C. Further, "oligonucleotides" includes modifications, known to
one skilled in the art, to the sugar backbone (e.g., ribose or
deoxyribose subunits), the sugar (e.g., 2' substitutions), the
base, and the 3' and 5' termini. The term "polynucleotide", as used
herein, has the same meaning as "oligonucleotide" is used
interchangeably with "polynucleotide".
[0028] Whenever an oligonucleotide is represented by a sequence of
letters, such as "ATGUCCTG," it will be understood that the
nucleotides are in 5'.fwdarw.3' order from left to right.
[0029] As used herein, "nucleoside" includes the natural
nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g. as
described in Komberg and Baker, DNA Replication, 2nd Ed. (Freeman,
San Francisco, 1992), and analogs. "Analogs" in reference to
nucleosides includes synthetic nucleosides having modified base
moieties and/or modified sugar moieties, e.g. described generally
by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such
analogs include synthetic nucleosides designed to enhance binding
properties, e.g. stability, specificity, or the like, such as
disclosed by Uhlmann and Peyman (Chemical Reviews, 90:543-584,
1990).
[0030] A "base" is defined herein to include (i) typical DNA and
RNA bases (uracil, thymine, adenine, guanine, and cytosine), and
(ii) modified bases or base analogs (e.g., 5-methyl-cytosine,
5-bromouracil, or inosine). A base analog is a chemical whose
molecular structure mimics that of a typical DNA or RNA base.
[0031] As used herein, "pyrimidine" means the pyrimidines occurring
in natural nucleosides, including cytosine, thymine, and uracil,
and common analogs thereof, such as those containing oxy, methyl,
propynyl, methoxy, hydroxyl, amino, thio, halo, and like,
substituents. The term as used herein further includes pyrimidines
with common protection groups attached, such as
N.sub.4-benzoylcytosine. Further common pyrimidine protection
groups are disclosed by Beaucage and Iyer (Tetrahedron 48:223-2311,
1992).
[0032] As used herein, "purine" means the purines occurring in
natural nucleosides, including adenine, guanine, and hypoxanthine,
and common analogs thereof, such as those containing oxy, methyl,
propynyl, methoxy, hydroxyl, amino, thio, halo, and like,
substituents. The term as used herein further includes purines with
common protection groups attached, such as N.sub.2-benzoylguanine,
N.sub.2-isobutyrylguanine, N.sub.6-benzoyladenine, and the like.
Further common purine protection groups are disclosed by Beaucage
and Iyer (cited above).
[0033] As used herein, the term "-protected-" as a component of a
chemical name refers to art-recognized protection groups for a
particular moiety of a compound, e.g. "5'-protected-hydroxyl" in
reference to a nucleoside includes triphenylmethyl (i.e., trityl),
p-anisyldiphenylmethyl (i.e., monomethoxytrityl or MMT),
di-p-anisylphenylmethyl (i.e., dimethoxytrityl or DMT), and the
like. Art-recognized protection groups include those described in
the following references: Gait, editor, Oligonucleotide Synthesis:
A Practical Approach (IRL Press, Oxford, 1984); Amarnath and Broom,
Chemical Reviews, 77:183-217, 1977; Pon et al., Biotechniques,
6:768-775, 1988; Ohtsuka et al., Nucleic Acids Research,
10:6553-6570, 1982; Eckstein, editor, Oligonucleotides and
Analogues: A Practical Approach (IRL Press, Oxford, 1991), Greene
and Wuts, Protective Groups in Organic Synthesis, Second Edition,
(John Wiley & Sons, New York, 1991), Narang, editor, Synthesis
and Applications of DNA and RNA (Academic Press, New York, 1987),
Beaucage and Iyer (cited above), and like references.
[0034] The term "halogen" or "halo" is used in its conventional
sense to refer to a chloro, bromo, fluoro or iodo substituent. In
the compounds described and claimed herein, halogen substituents
are generally fluoro, bromo, or chloro, preferably fluoro or
chloro.
[0035] The compounds of the present invention may be used to
inhibit or reduce telomerase enzyme activity and/or proliferation
of cells having telomerase activity. In these contexts, inhibition
or reduction of the enzyme activity or cell proliferation refer to
a lower level of the measured activity relative to a control
experiment in which the enzyme or cells are not treated with the
test compound. In particular embodiments, the inhibition or
reduction in the measured activity is at least a 10% reduction or
inhibition. One of skill in the art will appreciate that reduction
or inhibition of the measured activity of at least 20%, 50%, 75%,
90% or 100% may be preferred for particular applications.
[0036] The present invention is directed generally to
oligonucleotides containing at least one thiophosphoramidate
intersubunit linkage, methods of synthesizing such polynucleotides
and methods of using the inventive oligonucleotides as therapeutic
compounds and to in diagnostics.
[0037] The oligonucleotides are exemplified as having the formula:
##STR3##
[0038] wherein each B is independently selected to be a purine or
pyrimidine or an analog thereof such as uracil, thymine, adenine,
guanine, cytosine, 5-methylcytosine, 5-bromouracil and inosine,
[0039] Z.sub.1 is O or NH,
[0040] Z.sub.2 is OR, SR, or methyl wherein R is selected from the
group consisting of hydrogen, alkyl, aryl and salts thereof,
[0041] R.sub.1 is selected from the group consisting of hydrogen,
O--R.sub.2, S--R.sub.2, NHR.sub.2 and halogen, wherein R.sub.2 is
H, alkyl, or (CH.sub.2).sub.nW(CH.sub.2).sub.mH, where n is between
1-10, m is between 0-10 and W is O, S, or NH,
[0042] R.sub.3 and R.sub.4 are selected from the group consisting
of hydroxyl, amino and hydrogen, and
[0043] m.sub.2 is an integer between 1 and 50.
[0044] The nucleoside subunits making up the polynucleotides
nucleotides of the present invention can be selected to be in a
defined sequence: such as, a sequence of bases capable of
hybridizing specifically to a single-strand nucleic acid target
sequence or a sequence that will allow formation of a triplex
structure between the polynucleotide and a target duplex.
Preferably, the sequence of nucleoside subunits are joined by at
least one subunit that is a N3'.fwdarw.P5' thiophosphoramidate
defined by the formula: ##STR4##
[0045] wherein B is a purine or pyrimidine or an analog
thereof;
[0046] Z is OR, SR, or methyl, wherein R is selected from the group
consisting of hydrogen, alkyl, aryl and salts thereof; and
[0047] R.sub.1 is selected from the group consisting of hydrogen,
O--R.sub.2, S--R.sub.2, and halogen, wherein R.sub.2 is H, alkyl,
or (CH.sub.2).sub.nW(CH.sub.2).sub.mH, where n is between 1-10, m
is between 0-10 and W is O, S, or NH, with the proviso that when Z
is methyl or OMe, R.sub.1 is not H.
[0048] For example, all of the inter-subunit linkages of the
polynucleotide can be N3'.fwdarw.P'5 thiophosphoramidate
inter-subunit linkages defined by the formula: ##STR5##
[0049] The inventive oligonucleotides can be used to hybridize to
target nucleic acid sequences such as RNA and DNA. When desirable,
the oligonucleotides of the present invention can be labeled with a
reporter group, such as radioactive labels, biotin labels,
fluorescent labels and the like, to facilitate the detection of the
polynucleotide itself and its presence in, for example,
hybridization complexes.
[0050] In another aspect of the invention, a kit for isolating or
detecting a target RNA from a sample is provided. The kit contains
an oligonucleotide having a defined sequence of nucleoside subunits
joined by a least one intersubunit linkage defined by the formula:
##STR6##
[0051] where B is a purine or pyrimidine or an analog thereof; Z is
OR, SR, or methyl; and R.sub.1 is selected from the group
consisting of hydrogen, O--R.sub.2, S--R.sub.2, and halogen,
wherein R.sub.2 is H, alkyl, or (CH.sub.2).sub.nW(CH.sub.2).sub.mH,
where n is between 1-10, m is between 0-10 and W is O, S, or NH,
with the proviso that when Z is methyl or OMe, R.sub.1 is not H,
and
[0052] wherein the oligonucleotide hybridizes to the target
RNA.
[0053] The oligonucleotides can also be formulated as a
pharmaceutical inhibition of transcription or translation in a cell
in a disease condition related to overexpression of the target
gene.
[0054] Preferably, the sequence of nucleoside subunits are joined
by at least one inter-subunit linkage that is a N3'.fwdarw.P5'
thiophosphoramidate. Alternatively, all of the inter-subunit
linkages of the polynucleotide are N3'.fwdarw.P'5
thiophosphoramidate inter-subunit linkages defined by the formula:
##STR7##
[0055] In other aspects, the invention is directed to a solid phase
method of synthesizing oligonucleotide N3'.fwdarw.P5'
thiophosphoramidates using a modification of the phosphoramidite
transfer methodology of Nelson et al., (J. Organic Chemistry
62:7278-7287, 1997). The synthetic strategy employed
3'-NH-trityl-protected 3'-aminonucleoside
5'-O-cyanoethyl-N,N-diisopropylaminophosphoramidites (Nelson et
al., cited above) that were purchased from Cruachem and JBL
Scientific, Inc. (Aston, Pa. and San Luis Obispo, Calif.,
respectively). Every synthetic cycle (See FIG. 2) was conducted
using the following chemical procedures: 1) detritylation, 2)
coupling; 3) capping; 4) sulfurization. For a step-wise
sulfurization of the internucleaside phosphoramidite group formed
after the coupling step, the iodine/water based oxidizing agent was
replaced by the sulfurizing agents--either by elemental sulfur
S.sub.8 or by the commonly used Beaucage
reagent--3H-1,2-benzodithiol-3-one 1,1 dioxide (Iyer et al., J.
Organic Chemistry 55:4693-4699, 1990). The oligonucleotide
syntheses were performed (1 .mu.mole synthesis scale) with a 1%
solution of Beaucage reagent in anhydrous acetonitrile or 15%
S.sub.8 in CS.sub.2/Et.sub.3N, 99/1 (vol/vol) as the sulfurizing
agent.
[0056] Chimeric N3'.fwdarw.P5' phosphoramidate-phosphorthioamidate
oligonucleotides can be made by using an oxidation step(s) after
the coupling step, which results in formation of a phosphoramidate
internucleoside group. Similarly,
phosphodiester-phosphorthioamidates can be made by using
5'-phosphoramidite-3'-O-DMTr-protected nucleotides as monomeric
building blocks. These synthetic approaches are known in the
art.
[0057] The model phosphoramidate thymidine dinucleoside TnpsTn was
prepared using both types of sulfurizing agents and has a
3'-NHP(O)(S.sup.-)O-5' internucleoside group. The reaction mixtures
were analyzed and structure of the compound was confirmed by
ion-exchange (IE) and reverse phase (RP) HPLC, and .sup.31P NMR.
The analysis revealed that sulfurization of the internucleoside
phosphoramidite group with Beaucage reagent resulted in formation
of approximately 10-15% of the oxidized dinucleoside with
3'-NHP(O)(O.sup.-)O-5' phosphoramidate linkage (.sup.31P NMR
.delta., ppm 7.0 in D.sub.2O). Alternatively, sulfurization with
molecular sulfur S.sub.8 produced the desired dinucleotide
containing 3'-NHP(O)(S.sup.-)O-5' internucleoside group with
practically quantitative yield, as was judged by .sup.31P NMR and
IE HPLC analysis (.sup.31P NMR .delta., ppm 56.4, 59.6 in D.sub.2O,
Rp,Sp isomers).
[0058] Similar results with regards to the sulfurization efficiency
were obtained for the synthesis of model oligonucleotide 11-mer
GTTAGGGTTAG (SEQ ID NO:1), where sulfurization with Beaucage
reagent resulted in the full length product containing .about.15%
phosphoramidate linkages, as was judged by .sup.31P NMR analysis of
the reaction mixture. Chemical shifts for the main peaks were
.about.57 and 60 ppm (broad doublets) and 7 ppm (broad singlet)
corresponding to the thiophosphoramidate and phosphoramidate
groups, respectively. In contrast, sulfurization with S.sub.8
produced only .about.2% phosphoramidate linkages in the 11-mer
product according to the .sup.31P NMR analysis. The IE HPLC
analysis of the oligomer was in good agreement with the .sup.31P
NMR spectrum. Structure and purity of the final oligonucleotide
products was confirmed by MALDI-TOF mass spectra analysis, by
.sup.31P NMR, and by polyacrylamide gel electrophoretic analysis.
The molecular mass for thiophosphoramidate oligomers
GT.sub.2AG.sub.3T.sub.2AG (SEQ ID NO:1) and
TAG.sub.3T.sub.2AGACA.sub.2 (SEQ ID NO:2) was calculated to be
3,577.11 and 4,202.69, respectively. The molecular mass for
thiophosphoramidate oligomers GT.sub.2AG.sub.3T.sub.2AG (SEQ ID
NO:1) and TAG.sub.3T.sub.2AGACA.sub.2 (SEQ ID NO:2) was determined
experimentally by MALDI-TOF mass spectroscopy to be 3,577 and 4,203
respectively; mobility in 15% PAGE relative to isosequential
phosphoramidates was 0.95 and 0.97 respectively.
[0059] The model phosphoramidate nucleoside TnpsTn was
quantitatively converted into the phosphoramidate counterpart
TnpTn, by treatment with 0.1 M iodine solution in
pyridine/THF/H.sub.2O 1/4/0.1 (vol/vol), 55.degree. C., 15 min, as
judged by IE HPLC and .sup.31P NMR .sup.31P NMR .delta. ppm 7.0)
(See FIG. 3). Treatment of the TnpsTn dinucleotide with 10% acetic
acid, 55.degree. C., 48 hr unexpectedly resulted in only partial
hydrolysis (.about.10%) of internucleoside phosphoramidate linkage.
For comparison under these conditions the parent phosphoramidate
dimer TnpTn was completely hydrolyzed. Cleavage of the N--P bond in
the dinucleotide thiophosphoramidate was accompanied by concomitant
de-sulfurization process (.about.15%), followed by a rapid
hydrolysis of the resultant phosphoramidate --NHP(O)(O.sup.-)O--
group as revealed by IE HPLC and .sup.31P NMR (FIG. 3).
[0060] The 2'-R.sub.3 N3'.fwdarw.P5' thiophosphoramidates can be
obtained from the corresponding phophoramidates as described above.
The 2'-R.sub.3 N3'.fwdarw.P5' phosphoramidates were obtained by the
phosphoramidite transfer methodology devised for the synthesis of
oligonucleotide N3'.fwdarw.P5' phosphoramidates. The syntheisis of
2-O-alkyl N3'.fwdarw.P5' thiophosphoramidates is described in
detail as an illustration of this methodology.
[0061] The appropriately protected
2'-O-alkyl-3'aminonucleoside-5'-phosphoramidite building blocks 4,
6, 11, and 15, where alkyl is methyl, were prepared according to a
series of chemical transformations shown in Schemes 1-3 below. An
inventive step for the preparation of these compounds was the
selective methylation of the 2'-hybroxyl group in the presence of
either the imino functionality of pyrimidines, or the N-7 atom of
the purines. The two pyrimidine-based monomers were obtained from
the known
3-azido-2'-O-acetyl-5'-O-toluoyl-3'-deoxy-.beta.-D-ribofuranosyluracil
1. Typically, the N-3/O-4 imino nitrogen of 1 was first protected
with a protecting group, such as by the reaction of methyl
propyolate in the presence of dimethylaminopyridine (Scheme 1). The
crude reaction product was then selectively 2'-O-deacetylated, and
the resulting free 2'-hydroxyl group was then alkylated, such as by
methylation using iodomethane and silver oxide. The N-3 protecting
group was removed and the 3'-azido group was reduced to amine,
which was then immediately protected, such as reaction with
4-monomethoxytritylchloride, to give the precursor 3. The
5'-toluoyl ester was then cleaved using an alkaline solution,
followed by phosphitylation using known protocols to give the
desired 2'-O-methyl uridine phosphoramidite monomer 4. The
2'-O-methyl cytosine ##STR8## phosphoramidite was obtained by
conversion of uridine intermediate 3 into 3'-aminocytidine analogue
5.
[0062] The synthesis of the 2'-O-alkyl adenosine analogue required
the use of bulky protecting groups, primarily for exocyclic amine
in order to prevent the alkylation of N-7 during methylation of the
2'-hydroxyl group (Scheme 2).
3'-Azido-2'-O-acetyl-5'-O-toluoyl-N.sup.6-benzoyl-3'-deoxyadenosine
7 was first deprotected, such as by reaction with NH.sub.3/MeOH
(1/1, v/v), to afford 3'-azido-3'-deoxyadenosine. Then, the
5'-hydroxyl group and the N-6 moiety were selectively re-protected
with bulky protecting groups, such as the t-butyldiphenylsilyl
group or the 4-monomethoxytrityl group. The combination of the two
large substituents at the 5'-O and N-6 positions sterically
occluded N-7, thereby allowing for the selective introduction of a
methyl group at the 2'-position to produce the intermediate 8. The
N-6 4-15 monomethoxytrityl group was then removed, such as by
treatment with 3% trichloroacetic acid in an organic solvent, such
as dichloromethane, followed by re-protection of N-6. The use of
benzoyl chloride for the re-protection of N-6 resulted in the
addition of two benzoyl groups. The second benzoyl group was
subsequently removed by base treatment to produce the intermediate
9. The azide group was then reduced and the resulting 3'-amino
group was protected with 4-monomethoxytrityl to form 10. Finally,
the 5'-silyl protecting group was cleaved, and phosphitylation
resulted in the 2'-O-methyl phosporamidite monomer 11. ##STR9##
[0063] The synthesis of the guanosine-based 2'-O-alkyl
phosphramidite 15 is depicted in Scheme 3.
3'-Azido-2'-O-acetyl-5'-O-toluoyl-N.sup.2-isobutryl-O.sup.6-diphenylcarba-
moyl-3'-deoxyguanosine 12 was deblocked by treatment with a base.
The 5'O-- and O-6 were reprotected by reaction with
t-butyldiphenylsilylchloride. The bis-silylated intermediate was
then 2'-O alkylated. The O-6 silyl group was selectively
deprotected to give compound 13. The N-2 group was re-protected,
the 3'-azido group was reduced, and the resulting 3'-amino group
was protected to yield the nucleoside 14. Finally, the 2'-O-alkyl
guanosine phosphoramidite monomer 15 was obtained by removing the
5'-protecting group followed by phosphitylation of the unmasked
5'-hydroxyl. ##STR10##
[0064] In another embodiment of the present invention, the acid
stability of oligonucleotides is increased by placing subunits
linked by N3'.fwdarw.P5' thiophosphoramidate intersubunit linkages
in the oligonucleotides. The hybridization properties of the
thiophosphoramidate oligonucleotides were evaluated relative to
complementary DNA or RNA strands having phosphodiester or
phosphoramidate intersubunit linkages. The thermal stability data
for duplexes generated from phosphoramidate oligonucleotides and
phosphodiester oligomers are summarized in TABLE 1 (Example 3).
[0065] Hybridization of the thiophosphoramidate oligonucleotides
with complementary nucleic acids is sequence specific and
determined by the proper Watson-Crick base pairing. The duplex
formed by phosphoramidate oligonucleotide SEQ ID NO:3 with a single
base mismatch with a RNA target component of telomerase (Example 6,
TABLE 2, Experiment 2) is substantially less stable than the duplex
formed with Oligonucleotide SEQ ID NO:1 which is fully
complementary to the RNA component of telomerase (Example 6, TABLE
2, Experiment 1).
Applications of Oligonucleotides Containing Internucleoside 3'-NHP
(O) (S.sup.-) O-5' Thiophosphoramidate Linkages
[0066] Oligonucleotide SEQ ID NO:2 3'-NHP(O)(S.sup.-)O-5'
thiophosphoramidate was synthesized. This compound was surprisingly
acid stable and formed a stable complex with a complementary RNA
target. The N3'.fwdarw.P5' thiophosphoramidate polynucleotides of
the present invention have great potential for anti-sense and
anti-gene diagnostic/therapeutic applications. In a preferred
embodiment of the present invention, the oligonucleotides are
oligodeoxyribonucleotides.
[0067] A. Telomerase Inhibition Applications
[0068] Recently, an understanding of the mechanisms by which normal
cells reach the state of senescence, i.e., the loss of
proliferative capacity that cells normally undergo in the cellular
aging process, has begun to emerge. The DNA at the ends, or
telomeres, of the chromosomes of eukaryotes usually consists of
tandemly repeated simple sequences. Scientists have long known that
telomeres have an important biological role in maintaining
chromosome structure and function. More recently, scientists have
speculated that the cumulative loss of telomeric DNA over repeated
cell divisions may act as a trigger of cellular senescence and
aging, and that the regulation of telomerase, an enzyme involved in
the maintenance of telomere length, may have important biological
implications. See Harley, 1991, Mutation Research, 256:271-282.
Experiments by Bodnar et al. have confirmed the importance of
telomeres and telomerase in controlling the replicative lifespan of
cultured normal human cells. See Bodnar et al., 1998, Science
279:349-352.
[0069] Telomerase is a ribonucleoprotein enzyme that synthesizes
one strand of the telomeric DNA using as a template a sequence
contained within the RNA component of the enzyme. See Blackburn,
1992, Annu. Rev. Biochem., 61:113-129. The RNA component of human
telomerase has been sequenced and is 460 nucleotides in length
containing a series of 11-base sequence repeats that is
complementary to the telomere repeat. Human telomerase activity has
been inhibited by a variety of oligonucleotides complementary to
the RNA component of telomerase. See Norton et al., Nature
Biotechnology, 14:615, 1996; Pitts et al., Proc. Natl. Acad. Sci.,
95:11549-11554, 1998; and Glukhov et al., Bioch. Biophys. Res.
Commun., 248:368-371, 1999. Thiophosphoramidate oligonucleotides of
the present invention are complementary to 10 to 50 nucleotides of
telomerase RNA. Preferably, the inventive telomerase inhibitor
thiophosphoramidate oligonucleotides have a 10 to 20 consecutive
base sequence that is complementary to telomerase RNA.
[0070] Methods for detecting telomerase activity, as well as for
identifying compounds that regulate or affect telomerase activity,
together with methods for therapy and diagnosis of cellular
senescence and immortalization by controlling telomere length and
telomerase activity, have also been described. See, Feng, et al.,
1995, Science, 269:1236-1241; Kim, et al., 1994, Science,
266:2011-2014; PCT patent publication No. 93/23572, published Nov.
25, 1993; and U.S. Pat. Nos. 5,656,638, 5,760,062, 5,767,278,
5,770,613 and 5,863,936.
[0071] The identification of compounds that inhibit telomerase
activity provides important benefits to efforts at treating human
disease. Compounds that inhibit telomerase activity can be used to
treat telomerase-mediated disorders, such as cancer, since cancer
cells express telomerase activity and normal human somatic cells do
not possess telomerase activity at biologically relevant levels
(i.e., at levels sufficient to maintain telomere length over many
cell divisions). Unfortunately, few such compounds, especially
compounds with high potency or activity and compounds that are
bioavailable after oral administration, have been identified and
characterized. Hence, there remains a need for compounds that act
as telomerase inhibitors that have relatively high potency or
activity and that are orally bioavailable, and for compositions and
methods for treating cancer and other diseases in which telomerase
activity is present abnormally.
[0072] The new thiophosphoramidate oligonucleotide compounds of the
present invention are acid stable, and therefore, have many
valuable uses as inhibitors of deleterious telomerase activity,
such as, for example, in the treatment of cancer in humans.
Pharmaceutical compositions of thiophosphoramidate oligonucleotide
can be employed in treatment regimens in which cancer cells are
inhibited, in vivo, or can be used to inhibit cancer cells ex vivo.
Thus, this invention provides therapeutic compounds and
compositions for treating cancer, and methods for treating cancer
in mammals (e.g., cows, horses, sheep, steer, pigs and animals of
veterinary interest such as cats and dogs). In addition, the
phosphoramidate oligonucleotides of the present invention may also
be used to treat other telomerase-mediated conditions or diseases,
such as, for example, other hyperproliferative or autoimmune
disorders.
[0073] As noted above, the immortalization of cells involves inter
alia the activation of telomerase. More specifically, the
connection between telomerase activity and the ability of many
tumor cell lines to remain immortal has been demonstrated by
analysis of telomerase activity (Kim, et al., see above). This
analysis, supplemented by data that indicates that the shortening
of telomere length can provide the signal for replicative
senescence in normal cells, see PCT Application No. 93/23572,
demonstrates that inhibition of telomerase activity can be an
effective anti-cancer therapy. Thus, telomerase activity can
prevent the onset of otherwise normal replicative senescence by
preventing the normal reduction of telomere length and the
concurrent cessation of cell replication that occurs in normal
somatic cells after many cell divisions. In cancer cells, where the
malignant phenotype is due to loss of cell cycle or growth controls
or other genetic damage, an absence of telomerase activity permits
the loss of telomeric DNA during cell division, resulting in
chromosomal rearrangements and aberrations that lead ultimately to
cell death. However, in cancer cells having telomerase activity,
telomeric DNA is not lost during cell division, thereby allowing
the cancer cells to become immortal, leading to a terminal
prognosis for the patient. Agents capable of inhibiting telomerase
activity in tumor cells offer therapeutic benefits with respect to
a wide variety of cancers and other conditions (e.g., fungal
infections) in which immortalized cells having telomerase activity
are a factor in disease progression or in which inhibition of
telomerase activity is desired for treatment purposes. The
telomerase inhibitors of the invention can also be used to inhibit
telomerase activity in germ line cells, which may be useful for
contraceptive purposes.
[0074] In addition, it will be appreciated that therapeutic
benefits for treatment of cancer can be realized by combining a
telomerase inhibitor of the invention with other anti-cancer
agents, including other inhibitors of telomerase such as described
in U.S. Pat. Nos. 5,656,638, 5,760,062, 5,767,278, 5,770,613 and
5,863,936. The choice of such combinations will depend on various
factors including, but not limited to, the type of disease, the age
and general health of the patient, the aggressiveness of disease
progression, the TRF length and telomerase activity of the diseased
cells to be treated and the ability of the patient to tolerate the
agents that comprise the combination. For example, in cases where
tumor progression has reached an advanced state, it may be
advisable to combine a telomerase inhibiting compound of the
invention with other agents and therapeutic regimens that are
effective at reducing tumor size (e.g. radiation, surgery,
chemotherapy and/or hormonal treatments). In addition, in some
cases it may be advisable to combine a telomerase inhibiting agent
of the invention with one or more agents that treat the side
effects of a disease, e.g., an analgesic, or agents effective to
stimulate the patient's own immune response (e.g., colony
stimulating factor).
[0075] The compounds of the present invention demonstrate
inhibitory activity against telomerase activity in vivo, as can be
demonstrated as described below. The in vitro activities of the
compounds of the invention has also been demonstrated using the
methods described herein. As used herein, the term "in vitro"
refers to tests performed using living cells in tissue culture.
Such procedures are also known as "ex vivo".
[0076] Oligonucleotide telomerase inhibitors described in this
section typically comprise a sequence that is complementary to
telomerase RNA component. The sequence of human telomerase RNA
component is provided in U.S. Pat. No. 5,776,679. The telomerase
RNA component of other species can also be used, depending on the
intended subject of the therapy.
[0077] Generally, the oligonucleotide will comprise between about
10 and 100 nucleotides that are specific for telomerase (that is,
they hybridize with telomerase RNA component at lower
concentrations or under conditions of greater stringency than they
will with other RNA enzyme components, or other RNA molecules
expected to be present and functionally active in the target cells
or therapeutic bystander cells). Included are oligonucleotides
between about 10 and 25 nucleotides, exemplified by
oligonucleotides between 12 and 15 nucleotides, illustrated in the
Examples below. In many circumstances, the oligonucleotide will be
exactly complementary to a consecutive sequence of the same length
in telomerase RNA. Nevertheless, it is understood that
hybridization can still be specific even when there are mismatched
residues or gaps or additions in the oligonucleotide, especially
when the length of the corresponding complementary sequence in the
RNA is longer than 15 nucleotides.
[0078] One method used to identify thiophosphoramidate
polynucleotides of the invention with specific sequences that
inhibit telomerase activity involves placing cells, tissues, or
preferably a cellular extract or other preparation containing
telomerase in contact with several known concentrations of a
thiophosphoramidate oligonucleotide that is complementary to the
RNA component of telomerase in a buffer compatible with telomerase
activity. The level of telomerase activity for each concentration
of the thiophosphoramidate polynucleotide is measured. Before and
after administration of a telomerase inhibitor, telomerase activity
can be determined using standard reagents and methods. For example,
telomerase acvitity in cultured cells can be measured using TRAP
activity assay (Kim et al., Science 266:2011, 1997; Weinrich et
al., Nature Genetics 17:498, 1997). The following assay kits are
available commercially for research purposes: TRAPeze.RTM. XK
Telomerase Detection Kit (Cat. s7707; Intergen Co., Purchase
NY);.and TeloTAGGG Telomerase PCR ELISAplus (Cat. 2,013,89; Roche
Diagnostics, Indianapolis Ind.).
[0079] The IC.sub.50 (the concentration of the polynucleotide at
which the observed activity for a sample preparation is observed to
fall one-half of its original or a control value) for the
polynucleotide is determined using standard techniques. Other
methods for determining the inhibitory concentration of a compound
of the invention against telomerase can be employed as will be
apparent to those of skill in the art based on the disclosure
herein.
[0080] With the above-described methods, IC.sub.50 values for
several of the thiophosphoramidate oligonucleotides of the present
invention were determined, and found to be below 10 nM (See TABLE
2, Example 6).
[0081] With respect to the treatment of malignant diseases using
thiophosphoramidate polynucleotides that are complementary to the
RNA component of telomerase are expected to induce crisis in
telomerase-positive cell lines. Treatment of HME50-5E human breast
epithelial cells that were spontaneously immortalized with
thiophosphoramidate oligonucleotide SEQ ID NO:2 resulted in
inhibition of telomerase activity as demonstrated by the decrease
in telomere length (See Example 6, and FIG. 4). Treatment of other
telomerase-positive cell lines, such as HEK-293 and HeLa cells,
with inventive thiophosphoramidate oligonucleotides that are
complementary to the RNA sequence component of telomerase is also
expected to induce a reduction of telomere length in the treated
cells.
[0082] Thiophosphoramidate oligonucleotides of the invention are
also expected to induce telomere reduction during cell division in
human tumor cell lines, such as the ovarian tumor cell lines
OVCAR-5 and SK-OV-3. Importantly, however, in normal human cells
used as a control, such as BJ cells of fibroblast origin, the
observed reduction in telomere length is expected to be no
different from cells treated with a control substance, e.g., a
thiophosphoramidate oligonucleotide that has at least one single
base mismatch with the complementary telomerase RNA target. The
thiophosphoramidate oligonucleotides of the invention also are
expected to demonstrate no significant cytotoxic effects at
concentrations below about 20 .mu.M in the normal cells.
[0083] In addition, the specificity of the thiophosphoramidate
oligonucleotides of the present invention for telomerase RNA can be
determined by performing hybridization tests with and comparing
their activity (IC.sub.50) with respect to telomerase and to other
enzymes known to have essential RNA components, such as
ribonucleoase P. Compounds having lower IC.sub.50 values for
telomerase as compared to the IC.sub.50 values toward the other
enzymes being screened are said to possess specificity for
telomerase.
[0084] In vivo testing can also be performed using a mouse
xenograft model, for example, in which OVCAR-5 tumor cells are
grafted onto nude mice, in which mice treated with a
thiophosphoramidate oligonucleotide of the invention are expected
to have tumor masses that, on average, may increase for a period
following the initial dosing, but will begin to shrink in mass with
continuing treatment. In contrast, mice treated with a control
(e.g., a thiophosphoramidate oligonucleotide that has at least one
single base mismatch with the complementary telomerase RNA target)
are expected to have tumor masses that continue to increase.
[0085] From the foregoing those skilled in the art will appreciate
that the present invention also provides methods for selecting
treatment regimens involving administration of a
thiophosphoramidate oligonucleotide of the invention. For such
purposes, it may be helpful to perform a terminal restriction
fragment (TRF) analysis in which DNA from tumor cells is analyzed
by digestion with restriction enzymes specific for sequences other
than the telomeric (T.sub.2AG.sub.3).sub.N sequence. Following
digestion of the DNA, gel electrophoresis is performed to separate
the restriction fragments according to size. The separated
fragments are then probed with nucleic acid probes specific for
telomeric sequences to determine the lengths of the terminal
fragments containing the telomere DNA of the cells in the sample.
By measuring the length of telomeric DNA, one can estimate how long
a telomerase inhibitor should be administered and whether other
methods of therapy (e.g., surgery, chemotherapy and/or radiation)
should also be employed. In addition, during treatment, one can
test cells to determine whether a decrease in telomere length over
progressive cell divisions is occurring to demonstrate treatment
efficacy.
[0086] Thus, in one aspect, the present invention provides
compounds that can serve in the war against cancer as important
weapons against malignancies expressing telomerase, tumors
including skin, connective tissue, adipose, breast, lung, stomach,
pancreas, ovary, cervix, uterus, kidney, bladder, colon, prostate,
central nervous system (CNS), retina and circulating tumors (such
as leukemia and lymphoma). In particular, the thiophosphoramidate
polynucleotides of the present invention can provide a highly
general method of treating many, if not most, malignancies, as
demonstrated by the highly varied human tumor cell lines and tumors
having telomerase activity. More importantly, the
thiophosphoramidate oligonucleotides of the present invention can
be effective in providing treatments that discriminate between
malignant and normal cells to a high degree, avoiding many of the
deleterious side-effects present with most current chemotherapeutic
regimes which rely on agents that kill dividing cells
indiscriminately.
B. Other Antisense Applications
[0087] Antisense therapy involves the administration of exogenous
oligonucleotides that bind to a target nucleic acid, typically an
RNA molecule, located within cells. The term antisense is so given
because the oligonucleotides are typically complementary to mRNA
molecules ("sense strands") which encode a cellular product.
[0088] The thiophosphoramidate oligonucleotides described herein
are useful for antisense inhibition of gene expression (Matsukura
et al., Proc. Natl. Acad. Sci., 86:4244-4248, 1989; Agrawal et al.,
Proc. Natl. Acad. Sci., 86:7790-7794, 1989; Zamecnik et al., Proc.
Natl. Acad. Sci., 83:4143-4146, 1986; Rittner and Sczakiel, Nucleic
Acids Research, 19:1421-1426, 1991; Stein and Cheng, Science,
261:1004-1012, 1993). Oligonucleotides containing N3'.fwdarw.P5'
thiophosphoramidate linkages have therapeutic applications for a
large number of medically significant targets, including, but not
limited to inhibition of cancer cell proliferation and interference
with infectious viruses. The N3'.fwdarw.P5' thiophosphoramidate
oligonucleotides are useful for both veterinary and human
applications. The high acid stability of the inventive
oligonucleotides and their ability to act effectively as antisense
molecules at low concentrations (see below) make these
oligonucleotides highly desirable as therapeutic antisense
agents.
[0089] Anti-sense agents typically need to continuously bind all
target RNA molecules so as to inactivate them or alternatively
provide a substrate for endogenous ribonuclease H (Rnase H)
activity. Sensitivity of RNA/oligonucleotide complexes, generated
by the methods of the present invention, to Rnase H digestion can
be evaluated by standard methods (Donia, et al., J. Biol. Chem.,
268:14514-14522, 1993; Kawasaki, et al., J. Medicinal Chem.,
36:831-841, 1993).
[0090] The compounds and methods of the present invention provide
several advantages over the more conventional antisense agents.
First, thiophosphoramidate oligonucleotides bind more strongly to
RNA targets as corresponding phosphodiester oligonucleotides.
Second, the thiophosphoramidate oligonucleotides are more resistant
to degradation by acid conditions. Third, in cellular uptake of the
compound, an uncharged thiophosphoramidate polynucleotide backbone
may allow more efficient entry of the phosphoramidate
oligonucleotides into cells than a charged oligonucleotide.
[0091] Further, when an RNA is coded by a mostly purine strand of a
duplex target sequence, phosphoramidate analog oligonucleotides
targeted to the duplex also have potential for inactivating the
DNA--i.e., the ability to inactivate a pathogen in both
single-stranded and double-stranded forms (see discussion of
anti-gene therapies below).
[0092] Sequence-specific thiophosphoramidate oligonucleotide
molecules are potentially powerful therapeutics for essentially any
disease or condition that in some way involves RNA. Exemplary modes
by which such sequences can be targeted for therapeutic
applications include:
[0093] a) targeting RNA sequences expressing products involved in
the propagation and/or maintenance infectious agents, such as,
bacteria, viruses, yeast and other fungi, for example, a specific
mRNA encoded by an infectious agent;
[0094] b) formation of a duplex molecule that results in inducing
the cleavage of the RNA (e.g., Rnase H cleavage of RNA/DNA hybrid
duplex molecules);
[0095] c) blocking the interaction of a protein with an RNA
sequence (e.g., the interaction of TAT and TAR, see below); and
[0096] d) targeting sequences causing inappropriate expression or
proliferation of cellular genes: for example, genes associated with
cell cycle regulation; inflammatory processes; smooth muscle cell
(SMC) proliferation, migration and matrix formation (Liu, et al.,
Circulation, 79:1374-1387, 1989); certain genetic disorders; and
cancers (protooncogenes).
[0097] In one embodiment, translation or RNA processing of
inappropriately expressed cellular genes is blocked. Exemplary
potential target sequences are protooncogenes, for example,
including but not limited to the following: c-myc, c-myb, c-fos,
c-kit, ras, and BCR/ABL (e.g., Wickstrom, Editor, Prospects for
Antisense Nucleic Acid Therapy of Cancer and AIDS, Wiley-Liss, New
York, N.Y., 1991; Zalewski, et al., Circulation Res., 88:1190-1195,
1993; Calabretta, et al., Seminars in Cancer Biol., 3:391-398,
1992; Calabretta, et al., Cancer Treatment Rev. 19:169-179, 1993),
oncogenes (e.g., p53, Bayever, et al. Antisense Research and
Development, 3:383-390, 1993), transcription factors (e.g.,
NF.kappa.B, Cogswell, et al., J. Immunol., 150:2794-2804, 1993) and
viral genes (e.g., papillomaviruses, Cowsert, et al. Antimicrob.
Agents and Chemo., 37:171-177, 1993; herpes simplex virus, Kulka,
et al. Antiviral Res., 20:115-130, 1993). Another suitable target
for antisense therapy in hyperplasias is the protein component of
telomerase (see WO 99/50279), which is often the limiting component
in telomerase expression. The sequence of human telomerase reverse
transcriptase is provided in issued U.S. Pat. No. 6,093,809, in WO
98/14592, and in pGRN121 (ATCC Accession No. 209016). To further
illustrate, two RNA regions of the HIV-1 protein that can be
targeted by the methods of the present invention are the
REV-protein response element (RRE) and the TAT-protein
transactivation response element (TAR). REV activity requires the
presence of the REV response element (RRE), located in the HIV
envelope gene (Malim et al., Nature, 338:254-257, 1989; Malim et
al., Cell, 58:205-214, 1989).
[0098] The RRE has been mapped to a 234-nucleotide region thought
to form four stem-loop structures and one branched stem-loop
structure (Malim et al., Nature, 338:254-257, 1989). Data obtained
from footprinting studies (Holland et al., J. Virol., 64:5966-5975,
1990; Kjems et al., Proc. Natl. Acad. Sci., 88:683-687, 1991)
suggest that REV binds to six base pairs in one stem structure and
to three nucleotides in an adjacent stem-loop structure of the RRE.
A minimum REV binding region of about 40 nucleotides in stem-loop
II has been identified by Cook, et al. (Nucleic Acids Research,
19:1577-1583). This binding region can be target for generation of
RNA/DNA duplexes (e.g., Li, et al., J. Virol., 67:6882-6888, 1993)
using one or more thiophosphoramidate oligonucleotides, according
to the methods of the present invention.
[0099] The HIV-1 TAT is essential for viral replication and is a
potent transactivator of long terminal repeat (LTR)-directed viral
gene expression (Dayton et al., Cell, 44:941-947, 1986; Fisher et
al., Nature, 320:367-371, 1986). Transactivation induced by TAT
protein requires the presence of the TAR element (See U.S. Pat. No.
5,837,835) which is located in the untranslated 5' end of the viral
mRNA element.
[0100] The TAR element is capable of forming a stable stem-loop
structure (Muesing et al., Cell, 48:691-701, 1987). The integrity
of the stem and a 3 nucleotide (nt) bulge on the stem of TAR has
been demonstrated to be essential for specific and high-affinity
binding of the TAT protein to the TAR element (Roy et al., Genes
Dev., 4:1365-1373, 1990; Cordingley et al., Proc. Natl. Acad. Sci.,
87:8985-8989, 1990; Dingwall et al., Proc. Natl. Acad. Sci.,
86:6925-6929, 1989; Weeks et al., Science, 249:1281-1285, 1990).
This region can be targeted for anti-sense therapy following the
method of the present invention.
[0101] In addition to targeting the RNA binding sites of the REV,
RRE and TAT proteins, the RNA coding sequences for the REV and TAT
proteins themselves can be targeted in order to block expression of
the proteins.
[0102] Initial screening of N3'.fwdarw.P5' thiophosphoramidate
oligonucleotides, directed to bind potential antisense target
sites, typically includes testing for the thermal stability of
resultant RNA/DNA duplexes. When a thiophosphoramidate
oligonucleotide is identified that binds a selected RNA target
sequence, the oligonucleotide is further tested for inhibition of
RNA function in vitro. Cell culture assays systems are used for
such in vitro analysis (e.g., herpes simplex virus, Kulka, et al.
Antiviral Res., 20:115-130, 1993; HIV-1, Li, et al. J. Virol.,
67:6882-6888, 1993, Vickers, et al. Nucleic Acids Research,
19:3359-3368, 1991; coronary smooth muscle cell proliferation in
restenosis, Zalewski, et al. Nucleic Acids Research, 15:1699-1715,
1987; IL-2R, Grigoriev, et al. Proc. Natl. Acad. Sci.,
90:3501-3505, 1993; c-myb, Baer, et al. Blood, 79:1319-1326, 1992;
c-fos, Cutry, et al. J. Biol. Chem., 264:19700-19705, 1989;
BCR/ABL, Szczylik, et al., Science, 253:562-565, 1991).
C. Anti-Gene Applications
[0103] Inhibition of gene expression via triplex formation has been
previously demonstrated (Cooney et al., Science, 241:456-459, 1989;
Orson et al., Nucleic Acids Research, 19:3435-3441, 1991; Postel et
al., Proc. Natl. Acad. Sci., 88:8227-8231, 1991). The increased
stability of triplex structures formed when employing third strand
thiophosphoramidate analog oligonucleotides provides a stronger
tool for antigene applications, including veterinary and human
therapeutic applications.
[0104] A target region of choice is selected based on known
sequences using standard rules for triplex formation (Helene and
Toulme, Biochem. Biophys. Acta, 1049:99-125, 1990). Typically, the
thiophosphoramidate oligonucleotide sequence is targeted against
double-stranded genetic sequences in which one strand contains
predominantly purines and the other strand contains predominantly
pyrimidines.
[0105] Thiophosphoramidate oligonucleotides of the present
invention are tested for triplex formation against a selected
duplex target sequences using band shift assays (See for example,
U.S. Pat. No. 5,726,297, Example 4). Typically, high percentage
polyacrylamide gels are used for band-shift analysis and the levels
of denaturing conditions (Ausubel et al. Current Protocols in
Molecular Biology, Hohn Wiley and Sons, Inc. Media Pa.; Sauer et
al. Editor, Methods in Enzymology Protein/DNA Interactions,
Academic Press, 1991; Sambrook et al. In Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Vol. 2, 1989) are
adjusted to reduce any non-specific background binding.
[0106] The duplex target is labeled (for example, using a
radioactive nucleotide) and mixed with a third strand
oligonucleotide, being tested for its ability to form triplex
structures with the target duplex. A shift of the mobility of the
labeled duplex oligonucleotide indicates the ability of the
oligonucleotide to form triplex structures.
[0107] Triplex formation is indicated in the band shift assay by a
decreased mobility in the gel of the labeled triplex structure
relative to the labeled duplex structure.
[0108] Numerous potential target sites can be evaluated by this
method including target sites selected from a full range of DNA
sequences that vary in length as well as complexity.
Sequence-specific thiophosphoramidate analog binding molecules are
potentially powerful therapeutics for essentially any disease or
condition that in some way involves DNA. Exemplary target sequences
for such therapeutics include: a) DNA sequences involved in the
propagation and/or maintenance of infectious agents, such as,
bacterial, viruses, yeast and other fungi, for example, disrupting
the metabolism of an infectious agent; and b) sequences causing
inappropriate expression or proliferation of cellular genes, such
as oncogenes, for example, blocking or reducing the transcription
of inappropriately expressed cellular genes (such as genes
associated with certain genetic disorders).
[0109] Gene expression or replication can be blocked by generating
triplex structures in regions to which required regulatory proteins
(or molecules) are known to bind (for example, HIV transcription
associated factors like promoter initiation sites and SP1 binding
sites, McShan, et al., J. Biol. Chem., 267:5712-5721, 1992).
Alternatively, specific sequences within protein-coding regions of
genes (e.g., oncogenes) can be targeted as well.
[0110] When a thiophosphoramidate analog oligonucleotide is
identified that binds a selected duplex target sequence tests, for
example, by the gel band shift mobility assay described above, the
analog is further tested for its ability to form stable triplex
structures in vitro. Cell culture and in vivo assay systems, such
as those described U.S. Pat. No. 5,631,135, are used.
[0111] Target sites can be chosen in the control region of the
genes, e.g., in the transcription initiation site or binding
regions of regulatory proteins (Helene and Toulme, 1990; Birg et
al., 1990; Postel et al., 1991; Cooney et al., 1988). Also, target
sites can be chosen such that the target also exists in mRNA
sequences (i.e., a transcribed sequence), allowing oligonucleotides
directed against the site to function as antisense mediators as
well (see above).
[0112] Also, thiophosphoramidate modified DNA molecules can be used
to generate triplex molecules with a third strand target (i.e., a
single-strand nucleic acid). For example, a DNA molecule having two
regions capable of forming a triplex structure with a selected
target third strand molecule can be synthesized. Typically the two
regions are linked by a flexible region which allows the
association of the two regions with the third strand to form a
triplex.
[0113] Hinge regions can comprise any flexible linkage that keeps
the two triplex forming regions together and allows them to
associate with the third strand to form the triplex. Third strand
targets are selected to have appropriate purine/pyrimidine content
so as to allow formation of triplex molecules.
[0114] The flexible linkage may connect the two triplex forming
regions (typically, complementary DNA strands) in any selected
orientation depending on the nature of the base sequence of the
target. For example, the two triplex forming regions each have 5'
and 3' ends, these ends can be connected by the flexible hinge
region in the following orientations: 5' to 3', 3' to 5', 3' to 3',
and 5' to 5'.
[0115] Further, duplex DNA molecules containing at least one
thiophosphoramidate linkage in each strand can be used as decoy
molecules for transcription factors or DNA binding proteins (e.g.,
c-myb).
[0116] Single-stranded DNA can also be used as a target nucleic
acid for oligonucleotides of the present invention, using, for
example, thiophosphoramidate intersubunit linkage-containing
hairpin structures. Two thiophosphoramidate analog oligonucleotides
can be selected for single-strand DNA target-directed binding.
Binding of the two phosphoramidate analog strands to the
single-strand DNA target results in formation of a triplex.
D. Pharmaceutical Compositions
[0117] The present invention includes pharmaceutical compositions
useful in antisense and antigene therapies. The compositions
comprise an effective amount of N3'.fwdarw.P5' thiophosphoramidate
oligonucleotides in combination with a pharmaceutically acceptable
carrier. One or more N3'.fwdarw.P5' thiophosphoramidate
oligonucleotides (having different base sequences or linkages) may
be included in any given formulation.
[0118] The N3'.fwdarw.P5' thiophosphoramidate oligonucleotides,
when employed in therapeutic applications, can be formulated neat
or with the addition of a pharmaceutical carrier. The
pharmaceutical carrier may be solid or liquid. The formulation is
then administered in a therapeutically effective dose to a subject
in need thereof.
[0119] Liquid carriers can be used in the preparation of solutions,
emulsions, suspensions and pressurized compositions. The
N3'.fwdarw.P5' thiophosphoramidate oligonucleotides are dissolved
or suspended in a pharmaceutically acceptable liquid excipient.
Suitable examples of liquid carriers for parenteral administration
of N3'.fwdarw.P5' thiophosphoramidate oligonucleotides preparations
include water (partially containing additives, e.g., cellulose
derivatives, preferably sodium carboxymethyl cellulose solution),
alcohols (including monohydric alcohols and polyhydric alcohols,
e.g., glycols) and their derivatives, and oils (e.g., fractionated
coconut oil and arachis oil). The liquid carrier can contain other
suitable pharmaceutical additives including, but not limited to,
the following: solubilizers, suspending agents, emulsifiers,
buffers, thickening agents, colors, viscosity regulators,
preservatives, stabilizers and osmolarity regulators.
[0120] For parenteral administration of N3'.fwdarw.P5'
thiophosphoramidate oligonucleotides the carrier can also be an
oily ester such as ethyl oleate and isopropyl myristate. Sterile
carriers are useful in sterile liquid form compositions for
parenteral administration.
[0121] Sterile liquid pharmaceutical compositions, solutions or
suspensions can be utilized by, for example, intraperitoneal
injection, subcutaneous injection, intravenously, or topically. For
example, antisense oligonucleotides directed against retinal
cytomegalovirus infection may be administered topically by
eyedrops. N3'.fwdarw.P5' thiophosphoramidate oligonucleotides can
be also be administered intravascularly or via a vascular stent
impregnated with mycophenolic acid, for example, during balloon
catheterization to provide localized anti-restenosis effects
immediately following injury.
[0122] The liquid carrier for pressurized compositions can be
halogenated hydrocarbon or other pharmaceutically acceptable
propellant. Such pressurized compositions may also be lipid
encapsulated for delivery via inhalation. For administration by
intranasal or intrabronchial inhalation or insufflation,
N3'.fwdarw.P5' thiophosphoramidate oligonucleotides may be
formulated into an aqueous or partially aqueous solution, which can
then be utilized in the form of an aerosol, for example, for
treatment of infections of the lungs like Pneumocystis carnii.
[0123] N3'.fwdarw.P5' thiophosphoramidate oligonucleotides may be
administered topically as a solution, cream, or lotion, by
formulation with pharmaceutically acceptable vehicles containing
the active compound. For example, for the treatment of genital
warts.
[0124] The N3'.fwdarw.P5' thiophosphoramidate oligonucleotides may
be administered in liposome carriers. The use of liposomes to
facilitate cellular uptake is described, for example, in U.S. Pat.
No. 4,897,355 and U.S. Pat. No. 4,394,448. Numerous publications
describe the formulation and preparation of liposomes.
[0125] The dosage requirements for treatment with N3'.fwdarw.P5'
thiophosphoramidate oligonucleotides vary with the particular
compositions employed, the route of administration, the severity of
the symptoms presented, the form of N3'.fwdarw.P5'
thiophosphoramidate oligonucleotides and the particular subject
being treated.
[0126] For use as an active ingredient in a pharmaceutical
preparation, an oligonucleotide of this invention is generally
purified away from other reactive or potentially immunogenic
components present in the mixture in which they are prepared.
Typically, each active ingredient is provided in at least about 90%
homogeneity, and more preferably 95% or 99% homogeneity, as
determined by functional assay, chromatography, or gel
electrophoresis. The active ingredient is then compounded into a
medicament in accordance with generally accepted procedures for the
preparation of pharmaceutical preparations.
[0127] Pharmaceutical compositions of the invention can be
administered to a subject in a formulation and in an amount
effective to achieve any clinically desirable result. For the
treatment of cancer, desirable results include reduction in tumor
mass (as determined by palpation or imaging; e.g., by radiography,
CAT scan, or MRI), reduction in the rate of tumor growth, reduction
in the rate of metastasis formation (as determined e.g., by
histochemical analysis of biopsy specimens), reduction in
biochemical markers (including general markers such as ESR, and
tumor-specific markers such as serum PSA), and improvement in
quality of life (as determined by clinical assessment, e.g.,
Karnofsky score). For the treatment of viral infection, desirable
results include reduction or elimination of the infection, the
formation of infectious particles, or resolution of
disease-associated symptoms.
[0128] The amount of oligonucleotide per dose and the number of
doses required to achieve such effects can be determined
empirically using in vitro tests and animal models (illustrated in
Example 9). An appropriate range for testing can be estimated from
the 50% inhibitory concentration determined with isolated
telomerase or cultured cells. Preparations of isolated telomerase
can be obtained according to U.S. Pat. No. 5,968,506. Typically,
the formulation and route of administration will provide a local
concentration at the disease site of between 1 .mu.M and 1 nM for a
stable oligonucleotide of 12-15 nucleosides that is 100% identical
to an enzyme-specific target RNA sequence. The ultimate
responsibility for determining the administration protocol is in
the hands of the managing clinician.
[0129] In general, N3'.fwdarw.P5' thiophosphoramidate
oligonucleotides are administered at a concentration that affords
effective results without causing any harmful or deleterious side
effects (e.g., an effective amount). Such a concentration can be
achieved by administration of either a single unit dose, or by the
administration of the dose divided into convenient subunits at
suitable intervals throughout the day.
E. Diagnostic Applications
[0130] The thiophosphoramidate oligonucleotides of the present
invention are also useful in diagnostic assays for detection of RNA
or DNA having a given target sequence. In one general application,
the thiophosphoramidate oligonucleotides are labeled (e.g.,
isotopically or other detectable reporter group) and used as probes
for DNA or RNA samples that are bound to a solid support (e.g.,
nylon membranes).
[0131] Alternatively, the thiophosphoramidate oligonucleotides may
be bound to a solid support (for example, magnetic beads) and
homologous RNA or DNA molecules in a sample separated from other
components of the sample based on their hybridization to the
immobilized phosphoramidate analogs. Binding of thiophosphoramidate
oligonucleotides to a solid support can be carried out by
conventional methods. Presence of the bound RNA or DNA can be
detected by standard methods, for example, using a second labeled
reporter or polymerase chain reaction (See U.S. Pat. Nos. 4,683,195
and 4,683,202).
[0132] Diagnostic assays can be carried out according to standard
procedures, with suitable adjustment of the hybridization
conditions to allow thiophosphoramidate oligonucleotide
hybridization to the target region. The ability of
thiophosphoramidate oligonucleotides to bind at elevated
temperature can also help minimizes competition for binding to a
target sequence between the thiophosphoramidate oligonucleotides
probe and any corresponding single-strand phosphodiester
oligonucleotide that is present in the diagnostic sample.
[0133] Thiophorphoramidate oligonucleotides designed for use in
hybridization assays and other protocols described in this
disclosure can be packaged in kit form. The oligonucleotide is
provided in a container, typically in a buffer suitable for
long-term storage, and is optionally accompanied by other reagents,
standards, or controls useful in conducting the reaction.
Typically, the kit will also be accompanied by written indications
for use of the oligonucleotide in a hybridization reaction or
diagnostic assay, either as a product insert or by associated
literature in distribution or marketing of the kit.
F. Other Applications
[0134] In one aspect, the thiophosphoramidate oligonucleotides can
be used in methods to enhance isolation of RNA or DNA from samples.
For example, as discussed above, thiophosphoramidate
oligonucleotides can be fixed to a solid support and used to
isolate complementary nucleic acid sequences, for example,
purification of a specific mRNA from a polyA fraction (Goldberg, et
al. Methods in Enzmology, 68:206, 1979). The thiophosphoramidate
oligonucleotides are advantageous for such applications since they
can form more stable interactions with RNA and duplex DNA than
standard phosphodiester oligonucleotides.
[0135] A large number of applications in molecular biology can be
found for reporter labeled thiophosphoramidate oligonucleotides,
particularly for the detection of RNA in samples.
Thiophosphoramidate oligonucleotides can be labeled with
radioactive reporters (.sup.3H, .sup.14C, .sup.32P, or .sup.35S
nucleosides), biotin or fluorescent labels (Gryaznov, et al.,
Nucleic Acids Research, 20:3403-3409, 1992). Labeled
thiophosphoramidate oligonucleotides can be used as efficient
probes in, for example, RNA hybridization reactions (Ausubel, et
al., Current Protocols in Molecular Biology, Hohn Wiley and Sons,
Inc., Media, Pa.; Sambrook, et al., In Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Vol. 2,
1989).
[0136] Also, double-stranded DNA molecules where each strand
contains at least one thiophosphoramidate linkage can be used for
the isolation of DNA-duplex binding proteins. In this embodiment
the duplex containing thiophosphoramidate intersubunit linkages is
typically affixed to a solid support and sample containing a
suspected binding protein is then passed over the support under
buffer conditions that facilitate the binding of the protein to its
DNA target. The protein is typically eluted from the column by
changing buffer conditions.
[0137] The triplex forming DNA molecules described above,
containing thiophosphoramidate modified linkages, can be used as
diagnostic reagents as well, to, for example, detect the presence
of an DNA molecule in a sample.
[0138] Further, complexes containing oligonucleotides having
N3'.fwdarw.P5' thiophosphoramidate intersubunit linkages can be
used to screen for useful small molecules or binding proteins: for
example, N3'.fwdarw.P5' thiophosphoramidate oligonucleotide
complexes with duplex DNA can be used to screen for small molecules
capable of further stabilizing the triplex structure. Similar
screens are useful with N3'.fwdarw.P5' thiophosphoramidate
oligonucleotide complexes formed with single strand DNA and RNA
molecules.
G. Variations
[0139] Variations on the thiophosphoramidate oligonucleotides used
in the methods of the present invention include modifications to
facilitate uptake of the oligonucleotide by the cell (e.g., the
addition of a cholesterol moiety (Letsinger, U.S. Pat. No.
4,958,013); production of chimeric oligonucleotides using other
intersubunit linkages (Goodchild, Bioconjugate Chem., 1:165-187,
1990); modification with intercalating agents (for example, triplex
stabilizing intercalating agents, Wilson, et al., Biochemistry,
32:10614-10621, 1993); and use of ribose instead of deoxyribose
subunits.
[0140] Further modifications include, 5' and 3' terminal
modifications to the oligonucleotides (e.g., --OH, --OR, --NHR,
--NH.sub.2 and cholesterol). In addition, the ribose 2' position
can be the site of numerous modifications, including, but not
limited to, halogenation (e.g., --F).
[0141] N3'.fwdarw.P5' thiophosphoramidate oligonucleotides may also
be modified by conjugation to a polypeptide that is taken up by
specific cells. Such useful polypeptides include peptide hormones,
antigens and antibodies. For example, a polypeptide can be selected
that is specifically taken up by a neoplastic cell, resulting in
specific delivery of N3'.fwdarw.P5' thiophosphoramidate
oligonucleotides to that cell type. The polypeptide and
oligonucleotide can be coupled by means known in the art (see, for
example, PCT International Application Publication No.
PCT/US89/02363, WO8912110, published Dec. 14, 1989, Ramachandr, K.
et al.).
[0142] The properties of such modified thiophosphoramidate
oligonucleotides, when applied to the methods of the present
invention, can be determined by the methods described herein.
EXAMPLE 1
General Methods
[0143] .sup.31P NMR spectra were obtained on a Varian 400 Mhz
spectrometer. .sup.31P NMR spectra were referenced against 85%
aqueous phosphoric acid. Anion exchange HPLC was performed using a
Dionex DX 500 Chromatography System, with a Pharmacia Bitotech Mono
Q HR 5/5 or 10/16 ion exchange columns. Mass spectral analysis was
performed by Mass Consortium, San Diego, Calif. MALDI-TOF analysis
of oligonucleotides was obtained using a PerSpective Biosystems
Voyager Elite mass spectrometer with delayed extraction. Thermal
dissociation experiments were conducted on a Cary Bio 100 UV-Vis
spectrometer.
[0144] All reactions were carried out in oven dried glassware under
a nitrogen atmosphere unless otherwise stated. Commercially
available DNA synthesis reagents were purchased from Glen Research
(Sterling, Va.). Anhydrous pyridine, toluene, dichloromethane,
diisopropylethyl amine, triethylamine, acetic anhydride,
1,2-dichloroethane, and dioxane were purchased from Aldrich
(Milwaukee, Wis.).
[0145] All non-thiophosphoramidate oligonucleotides were
synthesized on an ABI 392 or 394 DNA synthesizer using standard
protocols for the phosphoramidite based coupling approach
(Caruthers, Acc. Chem. Res., 24:278-284, 1991). The chain assembly
cycle for the synthesis of oligonucleotide phosphoramidates was the
following: (i) detritylation, 3% trichloroaceticacid in
dichloromethane, 1 min; (ii) coupling, 0.1 M phosphoramidite and
0.45 M tetrazole in acetonitrile, 10 min; (iii) capping, 0.5 M
isobutyic anhydride in THF/lutidine, 1/1, v/v, 15 sec; and (iv)
oxidation, 0.1 M iodine in THF/pyridine/water, 10/10/1, v/v/v, 30
sec.
[0146] Chemical steps within the cycle were followed by
acetonitrile washing and flushing with dry argon for 0.2-0.4 min.
Cleavage from the support and removal of base and phosphoramidate
protecting groups was achieved by treatment with ammonia/EtOH, 3/1,
v/v, for 6 h at 55.degree. C. The oligonucleotides were
concentrated to dryness in vacuo after which the
2'-t-butyldimethylsilyl groups were removed by treatment with 1 M
TBAF in THF for 4-16 h at 25.degree. C. The reaction mixtures were
diluted with water and filtered through a 0.45 nylon acrodisc (from
Gelman Sciences, Ann Arbor, Mich.). Oligonucleotides were then
analyzed and purified by IE HPLC and finally desalted using gel
filtration on a Pharmacia NAP-5 or NAP-25 column. Gradient
conditions for IE HPLC: solvent A (10 mM NaOH), solvent B (10 mM
NaOH and 1.5 M NaCl); solvent A for 3 min then a linear gradient
0-80% solvent B within 50 min.
EXAMPLE 2
Synthesis of Arabino-fluorooligonucleotide N3'.fwdarw.P5'
Phosphoramidates
[0147] The solid phase synthesis of
oligo-2'-arabino-fluoronucleotide N3'.fwdarw.P5' phosphoramidates
was based on the phosphoramidite transfer reaction employing the
monomer building
blocks--5'-(O-cyanoethyl-N,N'-diisopropylamino)-phosphoramidites of
3'-MMTr-protected-3'-amino-2'-ara-fluoro nucleosides. Preparation
of the nucleoside monomers is depicted in Scheme 4. ##STR11##
[0148] Sugar precursor 1 (from Pfanstiehl) was converted into the
.alpha.-1-bromo intermediate 2 with retention of sugar C-1
configuration. Compound 2 was then used, without isolation, for a
S.sub.N2-type glycosylation reaction with silylated uracil and
thymine bases, which resulted in formation of nucleosides 3. Stereo
selectivity of the glycosylation reaction was quite high--more than
90% of the formed nucleoside 3 had the desired .beta.-anomeric
configuration, as was judged by .sup.1H NMR analysis of the
reaction mixture. The pure .beta.-isomer of nucleoside 3 was
isolated by crystallization from ethanol. Subsequently, 5'- and
3'-O-benzoyl protecting groups of 3 were removed in near
quantitative yields by treatment with methanolic ammonia. The
resultant 5'-,3'-hydroxyl groups containing nucleoside product was
then converted into the 2,3'-anhydronucleoside 4 under Mitsunobu
reaction conditions. The treatment of the 2,3'-anhydronuclesides
with lithium azide yielded the key 3'-azido precursor.quadrature.
5.quadrature.. This .quadrature. compound .quadrature. was then
converted into phosphoramidites 7t,u by the catalytic reduction of
3'-azido to 3'-amino group by hydrogenation, followed by
3'-tritylation, 5'-O-deprotection and 5'-O-phosphitylation.
Cytidine phosphoramidite 7c was obtained from the 3'-azido
precursor using the uracil-to-cytosine conversion process. Total
yields of the phosphoramidites 7c,t,u were in the range of 8-12%
based on the starting sugar precursor 1. Structure of the monomers
was confirmed by .sup.1H, .sup.31P, .sup.19F NMR and by mass
spectrometric analysis. Oligonucleotide synthesis using the
2'-arabino-fluoronucleotide monomer was then conducted on an
automated DNA/RNA ABI 394 synthesizer as described below.
EXAMPLE 3
Synthesis of Oligonucleotide N3'.fwdarw.P5'
Thiophosphoramidates
[0149] Oligonucleotide N3'.fwdarw.P5' thiophosphoramidates were
prepared by the amidite transfer reaction on an ABI 394
synthesizer. The fully protected monomer building blocks were
3'-aminotrityl nucleoside-5'-(2-cyanoethyl-N,N-diisopropyl)
phosphoramidite where nucleoside is 3'-deoxy-thymidine,
2',3'-dideoxy-N.sup.2-isobutyryl-guanosine,
2',3'-dideoxy-N.sup.6-benzoyl-adenosine or
2',3'-dideoxy-N-benzoyl-cytidine.
5'-Succinyl-3'-aminotrityl-2',3'-dideoxy nucleosides were coupled
with an amino group containing long chain controlled pore glass
(LCAA-CPG) and used as the solid support. The synthesis was
performed in the direction of 5' to 3'. The following protocol was
used for the assembly of oligonucleotide N3'.fwdarw.P5'
thiophosphoramidates: (i) detritylation, 3% dichloroacetic acid in
dichloromethane; (ii) coupling, 0.1 M phosphoramidite and 0.45 M
tetrazole in acetonitrile, 25 sec; (iii) capping, isobutyric
anhydride/2,6-lutidine/THF 1/1/8 v/v/v as Cap A and standard Cap B
solution; (iv) sulfurization, 15% S.sub.8 in carbon disulfide
containing 1% triethylamine, 1 min. Before and after the
sulfurization step the column was washed with neat carbon disulfide
to prevent elemental sulfur precipitation. The oligonucleotide
thiophosphoramidates were cleaved from the solid support and
deprotected with concentrated aqueous ammonia. The compounds were
analyzed and purified by HPLC. Ion exchange (IE) HPLC was performed
using DIONEX DNAPac.TM. ion exchange column at pH 12 (10 mM NaOH)
with a 1%/min linear gradient of 10 mM NaOH in 1.5 M NaCl and a
flow rate of 1 ml/min. The products were desalted on Sephadex NAP-5
gel filtration columns (Pharmacia) and lyophilized in vacuo.
.sup.31P NMR experiments were performed in deuterium oxide to
analyze the extent of sulfurization analysis (.sup.31P NMR .delta.,
ppm 58, 60 broad signals Rp,Sp isomers).
[0150] Oligonucleotide thiophosphoramidate 5'-GTTAGGGTTAG-3' (SEQ
ID NO:1) was synthesized the following way: An ABI Model 394
synthesizer was set up with 0.1 M solutions of
3'-tritylamino-2',3'-dideoxy-N.sup.6-benzoyl-adenosine
(N.sup.2-isobutyryl-guanosine, and thymidine)
5'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites. The reagent
bottle of station #10 was filled with neat carbon disulfide and
reagent bottle #15 was filled with a solution of 15% S.sub.8 in
carbon disulfide containing 1% triethylamine. As the activator the
commercially available 0.45 M solution of tetrazole in acetonitrile
was used. Cap A solution (station #11) was replaced by
tetrahydrofuran/isobutyric anhydride/2,6-lutidine 8/1/1 v/v/v
solution. Cap B was also the commercially available reagent. A new
function was created to deliver carbon disulfide from station #10
to the column. The default sulfur synthesis cycle was modified the
following way: sulfurization time was set at 1 min., and before and
after sulfurization carbon disulfide was delivered to the column
for 20 s. The synthesis column was filled with 1 .mu.mole solid
support
N.sup.2-isobutyryl-3'-(trityl)amino-2',3'-dideoxyguanosine-5'-succinyl-lo-
aded CPG (controlled pore glass). The sequence of the compound was
programmed as GATTGGGATTG (5'.fwdarw.3') (SEQ ID NO:11). The trityl
group was removed at end of the synthesis and the column was washed
manually with carbon disulfide and acetonitrile. The solid support
was removed from the column and treated with 1 ml concentrated
aqueous ammonia at 55.degree. C. for 6 hr in a tightly closed glass
vial. After filtration most of the ammonia was evaporated and the
remaining solution was desalted using Sephadex.TM. NAP-5 gel
filtration columns (Pharmacia) followed by lyophilization in vacuo.
The product was analyzed and purified as described above. All of
the other thiophosphoramidate oligonucleotides listed in Table 2
(SEQ ID NO:2-4) were synthesized using the above described
methods.
EXAMPLE 4
Acid Stability and Duplex Formation Properties of Oligonucleotide
N3'.fwdarw.P5' Thiophosphoramidates
[0151] Oligonucleotide thiophosphoramidates unexpectedly
demonstrated an increased acid stability relative to
phosphoramidate counterparts. One might have expected that
substitution of the non-bridge oxygen of the internucleotide
phosphate group with sulfur should have resulted in a decrease in
the acid stability of the phoshoramidate because the difference in
the electron-donating properties of sulfur verses oxygen, which
could have made the protonation of the 3'-NH easier. However,
contrary to this prediction, the thiophosphoramidate
internucleotide linkages were found to be more acid stable than
their oxo-phosphoramidate counterparts.
[0152] The half-lives of thiophosphoramidate
TAG.sub.3T.sub.2AGACA.sub.2 (SEQ ID NO:2) and its phosphoramidate
counterpart in 40% aqueous acetic acid at room temperature were
approximately 6 hours and 0.5 hour, respectively, according to IE
HPLC analysis (See Table 1). Moreover, the composition of the
hydrolysis products was different between the thiophosphoramidate
and its phosphoramidate counterpart. The acid hydrolysis of the
thiophosphoramidate appears to initially result in
de-sulfurization, rather than cleavage of internucleoside N--P
groups, as it occurs for the phosphoramidates. These results
indicated a much higher resistance to acidic conditions of the
thiophosphoramidates than that of the phosphoramidate
oligonucleotides, thus indicating that this new class of
thiophosphoramidate oligonucleotides has improved potential for the
development of oral oligonucleotide therapeutics as compared to
other phosphoramidate oligonucleotides. TABLE-US-00001 TABLE 1 Tm,
Acid Expt Oligomer Type.sup.a .degree. C..sup.b -Tm, .degree.
C..sup.c Stability.sup.d 1. GTTAGGGTTAG po 44.2 -- SEQ ID NO: 1 2.
TAGGGTTAGACAA po 45.2 -- SEQ ID NO: 2 3. Same as expt 1 np 72.1
27.9 4. Same as expt 2 np 71.7 26.5 0.5 hr 5. Same as expt 1 nps
71.5 27.3 6. Same as expt 2 nps 70.0 24.8 6 hr .sup.apo, np, nps
correspond to phosphodiester, N3'.fwdarw.P5' phosphoramidate and
thiophosphoramidate groups, respectively; .sup.bmelting
temperature, Tm (.+-.0.5.degree. C.) of the duplexes formed with a
complementary natural RNA oligomer in 150 mM NaCl, 10 mM sodium
phosphate buffer pH 7.4; .sup.cincrease of Tm relative to the
natural phosphodiester counterpart; .sup.dhalf-live of
oligonucleotide in 40% aqueous acetic acid at room temperature.
[0153] Duplex formation properties of oligonucleotide
phosphoramidates with complementary RNA strand were evaluated using
thermal dissociation experiments. The results are summarized in
Table 1. The presented data show that the oligonucleotide
thiophosphoramidates formed significantly more stable complexes
than the isosequential natural phosphodiester oligomers wherein the
difference in Tm was .about.25-27.degree. C. per oligomer. Also,
the increase in the thermal stability of duplexes was similar to
that observed for the phosphoramidate oligomers. This indicates
that the substitution of non-bridging oxygen by sulfur atom in
internucleoside phosphoramidate group did not alter RNA binding
properties of these compounds significantly, which was determined
by N-type sugar puckering of the 3'-aminonucleosides and by
increased sugar-phosphate backbone hydration.
EXAMPLE 5
Preparation of Affinity Purified Extract Having Telomerase
Activity
[0154] Extracts used for screening telomerase inhibitors were
routinely prepared from 293 cells over-expressing the protein
catalytic subunit of telomerase (hTERT). These cells were found to
have 2-5 fold more telomerase activity than parental 293 cells. 200
ml of packed cells (harvested from about 100 liters of culture)
were resuspended in an equal volume of hypotonic buffer (10 mM
Hepes pH 7.9, 1 mM MgCl.sub.2, 1 mM DTT, 20 mM KCl, 1 mM PMSF) and
lysed using a dounce homogenizer. The glycerol concentration was
adjusted to 10% and NaCl was slowly added to give a final
concentration of 0.3 M. The lysed cells were stirred for 30 min and
then pelleted at 100,000.times.g for 1 hr. Solid ammonium sulfate
was added to the S100 supernatant to reach 42% saturation. The
material was centrifuged; the pellet was resuspended in one fifth
of the original volume and dialyzed against Buffer `A` containing
50 mM NaCl. After dialysis the extract was centrifuged for 30 min
at 25,000.times.g. Prior to affinity chromatography, Triton
X-100.TM. (0.5%), KCl (0.3 M) and tRNA (50 .mu.g/ml) were added.
Affinity oligo (5' biotinTEG-biotinTEG-biotinTEG-GTA GAC CTG TTA
CCA guu agg guu ag 3' [SEQ ID NO:5]; lower case represents 2'
O-methyl ribonucleotides and upper case represents
deoxynucleotides) was added to the extract (1 nmol per 10 ml of
extract). After an incubation of 10 min at 30.degree. C.,
Neutravidin beads (Pierce; 250 .mu.l of a 50% suspension) were
added and the mixture was rotated overnight at 4.degree. C. The
beads were pelleted and washed three times with Buffer `B`
containing 0.3 M KCl, twice with Buffer `B` containing 0.6 M KCl,
and twice more with Buffer B containing 0.3 M KCl. Telomerase was
eluted in Buffer `B` containing 0.3 M KCl, 0.15% Triton X-100.TM.
and a 2.5 molar excess of displacement oligo (5'-CTA ACC CTA ACT
GGT AAC AGG TCT AC-3' [SEQ ID NO:6] at 0.5 ml per 125 .mu.l of
packed Neutravidin beads) for 30 min. at room temperature. A second
elution was performed and pooled with the first. Purified extracts
typically had specific activities of 10 fmol nucleotides
incorporated/min/.mu.l extract, or 200 nucleotides/min/mg total
protein. TABLE-US-00002 Buffer `A` Buffer `B` 20 mM Hepes pH 7.9 20
mM Hepes pH 7.9 1 mM MgCl2 1 mM EDTA 1 mM DTT 1 mM DTT 1 mM EGTA
10% glycerol 10% glycerol 0.5 Triton X-100 .TM.
EXAMPLE 6
Telomerase Inhibition by Oligonucleotide N3'.fwdarw.P5'
Thiophosphoramidates
[0155] Three separate 100 .mu.l telomerase assays are set up with
the following buffer solutions: 50 mM Tris acetate, pH 8.2, 1 mM
DTT, 1 mM EGTA, 1 mM MgCl.sub.2, 100 mM K acetate, 500 .mu.M dATP,
500 .mu.M TTP, 10 .mu.M [.sup.32P-]dGTP (25 Ci/mmol), and 100 nM
d(TTAGGG).sub.3 [SEQ ID NO:7]. To the individual reactions 2.5, 5
or 10 .mu.l of affinity-purified telomerase (see Example 4) is
added and the reactions are incubated at 37.degree. C. At 45 and 90
minutes, 40 .mu.l aliquots are removed from each reaction and added
to 160 .mu.l of Stop Buffer (100 mM NaCl, 10 mM Na pyrophosphate,
0.2% SDS, 2 mM EDTA, 100 .mu.g/ml tRNA). 10 .mu.l trichloroacetic
acid (TCA) (100%) is added and the sample is incubated on ice for
30 minutes. The sample is pelleted in a microcentrifuge
(12000.times.g force) for 15 minutes. The pellet is washed with 1
ml 95% ethanol and pelleted again in the microcentrifuge
(12000.times.g force) for 5 minutes. The pellet is resuspended in
50 .mu.l dH.sub.2O and transferred to a 12.times.75 glass test tube
containing 2.5 ml of ice cold solution of 5% TCA and 10 mM Na
pyrophosphate. The sample is incubated on ice for 30 minutes. The
sample is filtered through a 2.5 cm wet (dH.sub.2O) GFC membrane
(S&S) on a vacuum filtration manifold. The filter is washed
three times under vacuum with 5 ml ice cold 1% TCA, and once with 5
ml 95% ethanol. The filter is dried and counted in a scintillation
counter using scintillation fluid. The fmol of nucleotide
incorporated is determined from the specific activity of
radioactive tracer. The activity of extract is calculated based on
the dNTP incorporated and is expressed as fmol dNTP/min/.mu.l
extract.
Telomerase Activity Assay
Bio-Tel FlashPlate Assay
[0156] An assay is provided for the detection and/or measurement of
telomerase activity by measuring the addition of TTAGGG telomeric
repeats to a biotinylated telomerase substrate primer; a reaction
catalyzed by telomerase. The biotinylated products are captured in
streptavidin-coated microtiter plates. An oligonucleotide probe
complementary to 3.5 telomere repeats labeled with .sup.33P is used
for measuring telomerase products, as described below. Unbound
probe is removed by washing and the amount of probe annealing to
the captured telomerase products is determined by scintillation
counting.
Method:
[0157] Thiophosphoramidate oligonucleotides were stored as
concentrated stocks and dissolved in PBS.
[0158] For testing, the thiophosphoramidate oligonucleotides were
diluted to a 15.times. working stock in PBS and 2 .mu.l was
dispensed into two wells of a 96-well microtiter dish (assayed in
duplicate).
[0159] Telomerase extract was diluted to a specific activity of
0.04-0.09 fmol dNTP incorporated/min./.mu.l in Telomerase Dilution
Buffer and 18 .mu.l added to each sample well to preincubate with
compound for 30 minutes at room temperature.
[0160] The telomerase reaction was initiated by addition of 10
.mu.l Master Mix to the wells containing telomerase extract and
oligonucleotide compound being tested. The plates were sealed and
incubated at 37.degree. C. for 90 min.
[0161] The reaction was stopped by the addition of 10 .mu.l
HCS.
[0162] 25 .mu.l of the reaction mixture was transferred to a
96-well streptavidin-coated FlashPlate.TM. (NEN) and incubated for
2 hours at room temperature with mild agitation.
[0163] The wells were washed three times with 180 .mu.l 2.times.SSC
without any incubation.
[0164] The amount of probe annealed to biotinylated telomerase
products were detected in a scintillation counter.
Buffers:
Telomerase Dilution Buffer
[0165] 50 mM Tris-acetate, pH 8.2
[0166] 1 mM DTT
[0167] 1 mM EGTA
[0168] 1 mM MgCl.sub.2
[0169] 830 nM BSA
Master Mix (MM)
[0170] 50 mM Tris-acetate, pH 8.2
[0171] 1 mM DTT
[0172] 1 mM EGTA
[0173] 1 mM MgCl.sub.2
[0174] 150 mM K acetate
[0175] 10 .mu.M dATP
[0176] 20 .mu.M dGTP
[0177] 120 .mu.M dTTP
[0178] 100 nM biotinylated primer (5'-biotin-AATCCGTCGAGCAGAGTT-3')
[SEQ ID NO:8]
[0179] 5.4 nM labeled probe [5'-CCCTAACCCTAACCCTAACCC-(.sup.33P)
A.sub.1-50-3'] [SEQ ID NO:9]; specific activity approximately
10.sup.9 cpm/.mu.g or higher
Hybridization Capture Solution (HCS)
[0180] 12.times.SSC (1.times.=150 mM NaCl/30 mM
Na.sub.3Citrate)
[0181] 40 mM EDTA
[0182] 40 mM Tris-HCl, pH 7.0
[0183] Using the foregoing assay, the thiophosphoramidate
oligonucleotides represented by SEQ ID NO:1 and SEQ ID NO:2 were
shown to have telomerase IC.sub.50 values below 1.0 nM (See TABLE
2, experiments 1 and 3). TABLE-US-00003 TABLE 2 EVALUATION OF
OLIGOS 1-4 AS TELOMERASE INHIBITORS IN COMPARISON WITH
PHOSPHORAMIDATES: IC.sub.50(nM) IC.sub.50(nM) thio- EXP
Oligonucleotide phosphoramidate phosphoramidate 20.
5'-GTTAGGGTTAG-3' 1.9 0.89 SEQ ID NO: 1 21. 5'-GTTGAGTGTAG-3' 1000
177.4 SEQ ID NO: 3 22. 5'-TAGGGTTAGACAA-3' 1.64 0.41 SEQ ID NO: 2
23. 5'-TAGGTGTAAGCAA-3' 1000 79.3 SEQ ID NO: 4
[0184] Oligonucleotide sequence 2 (SEQ ID NO:3) is a mismatch
control for the oligonucleotides used in experiment 1 (SEQ ID
NO:1). Similarly, oligonucleotide sequence 4 (SEQ ID NO:4) is a
mismatch control for the oligonucleotides (SEQ ID NO:2). used in
experiment 3.
[0185] The telomerase inhibition data presented in Table 2 show
that the thiophosphoramidate polynucleotides of the present
invention are about 2-3 times better at inhibiting telomerase
activity relative to counterpart phosphoramidates oligonucleotides.
Thus, the inventive thiophosphoramidate oligonucleotides are not
only more active in the telomerase inhibition assay, as compared to
their phosphoramidate counterparts, but are also more acid
resistant than them as well. This combination of characteristics
imparts the inventive thiophosphoramidate oligonucleotides with an
important advantage compared to phosphoramidate
polynucleotides.
EXAMPLE 7
Anti-tumor Activity of Thiophosphoramidate Oligonucleotides
Ex vivo Studies
[0186] a. Reduction of Telomere Length in Tumor Cells
[0187] Colonies of human breast epithelial cells (spontaneously
immortalized) were prepared using standard methods and materials.
Colonies were prepared by seeding 15-centimeter dishes with about
10.sup.6 cells in each dish. The dishes were incubated to allow the
cell colonies to grow to about 80% confluence, at which time each
of the colonies were divided into two groups. One group was exposed
to a subacute dose of thiophosphoramidate polynucleotide SEQ ID
NO:2 at a predetermined concentration (e.g., between about 100 nM
and about 20 .mu.M) for a period of about 4-8 hours after plating
following the split. The second group of cells were similarly
exposed to mismatch control oligonucleotide SEQ ID NO:4.
[0188] Each group of cells is then allowed to continue to divide,
and the groups are split evenly again (near confluence). The same
number of cells were seeded for continued growth. The test
thiophosphoramidate oligonucleotide or control oligonucleotide was
added every fourth day to the samples at the same concentration
delivered initially. In one experiment the cells were additionally
treated with FuGENE6.TM. (Boehringer-Mannhiem) following
manufacturers instructions. FuGENE6.TM. enhances oligonucleotide
uptake by the cells.
[0189] Telomere length was determined by digesting the DNA of the
cell samples using restriction enzymes specific for sequences other
than the repetitive T.sub.2AG.sub.3 sequence of human telomeres
(TRF analysis). The digested DNA was separated by size using
standard techniques of gel electrophoresis to determine the lengths
of the telomeric repeats, which appear, after probing with a
telomere DNA probe, on the gel as a smear of high-molecular weight
DNA (approximately 2 Kb-15 Kb). FIGS. 4 and 5 show examples of such
experiments.
[0190] The results presented in FIG. 4 indicate that the
thiophosphoramidate oligonucleotide SEQ ID NO:2 is a potent in
vitro inhibitor of telomerase activity. In the absence of
FuGENE6.TM., the thiophoshoramidate oligonucleotide SEQ ID NO:2
induced a large decrease in telomere length when incubated with
HME50-5E cells in the range of 1-20 .mu.M. When cells were
coincubated with FuGENE6 and thiophoshoramidate oligonucleotide SEQ
ID NO:2 telomere size was reduced compared to the control cells at
even the lowest concentration tested (100 nM).
[0191] The results presented in FIG. 5 indicate that the
thiophoshoramidate oligonucleotide having the sequence
CAGTTAGGGTTAG (SEQ ID NO:10) is a potent in vitro inhibitor of
telomerase activity. When the cells were incubated with the
thiophoshoramidate oligonucleotide SEQ ID NO:10, telomere size was
reduced compared to the control cells at even the lowest
concentration tested (1 nM). Thus, the inventive
thiophosphoramidate oligonucleotides are potent in vitro inhibitors
of telomerase activity in immortalized human breast epithelial
cell.
[0192] In another experiment, HME50-5E cells were incubated with
one of the thiophosphoramidate polynucleotides shown in SEQ ID NOs:
2 and 10. The mismatch oligonucleotide SEQ ID NO:4 was used as a
control. All polynucleotides were used at concentrations between
about 0.1 .mu.M and about 20 .mu.M using the protocol described
above. The data on cell growth, shown in FIG. 6, indicates that the
cell entered crisis (i.e., the cessation of cell function) within
about 100 days following administration of the test
thiophosphoramidate oligonucleotides of the invention.
[0193] In addition, TRF analysis of the cells (FIG. 7) using
standard methodology shows that the test thiophosphoramidate
oligonucleotides of the invention were effective in reducing
telomere length. The HME50-5E cells were incubated with one of the
thiophosphoramidate polynucleotides shown in SEQ ID NOs: 2 and 10.
The mismatch oligonucleotide SEQ ID NO:4 was used as a control. All
polynucleotides were used at a concentration of about 0.5 .mu.M
using the protocol described above. The length of the telomeres
were measured at 10, 20, 40 and 80 days. For the cells with the
control mismatch oligonucleotide, the telomere lenght was measured
at day 90, and this data point served as the end point. In addition
to the HME50-5E cells described above, this assay can be performed
with any telomerase-positive cell line, such as HeLa cells or
HEK-293 cells.
[0194] b. Specificity
[0195] Thiophosphoramidate polynucleotides of the invention are
screened for activity (IC.sub.50) against telomerase and other
enzymes known to have RNA components by performing hybridization
tests or enzyme inhibition assays using standard techniques.
Oligonucleotides having lower IC.sub.50 values for telomerase as
compared to the IC.sub.50 values toward the other enzymes being
screened are said to possess specificity for telomerase.
[0196] c. Cytotoxicity
[0197] The cell death (XTT) assay for cytotoxicity was performed
using HME50-5E, Caki-1, A431, ACHN, and A549 cell types. The cell
lines used in the assay were exposed to the thiophosphoramidate
oligonucleotide of SEQ ID NO:2 for 72 hours at concentrations
ranging from about 1 .mu.M to about 100 .mu.M in the presence and
absence of lipids. During this period, the optical density (OD) of
the samples was determined for light at 540 nanometers (nm). The
IC.sub.50 values obtained for the various cell types (shown in FIG.
8) were generally less than 1 .mu.M. Thus, no significant cytotoxic
effects are expected to be observed at concentrations less than
about 100 .mu.M. It will be appreciated that other tumor cells
lines such as the ovarian tumor cell lines OVCAR-5 and SK-OV-3 can
be used to determine cytotoxicity in addition to control cell lines
such as normal human BJ cells. Other assays for cytotoxicity such
as the MTT assay (see Berridge et al., Biochemica 4:14-19, 1996)
and the alamarBlue.TM. assay (U.S. Pat. No. 5,501,959) can be used
as well.
[0198] Preferably, to observe any telomerase inhibiting effects the
thiophosphoramidate oligonucleotides should be administered at a
concentration below the level of cytotoxicity. Nevertheless, since
the effectiveness of many cancer chemotherapeutics derives from
their cytotoxic effects, it is within the scope of the present
invention that the thiophosphoramidate oligonucleotides of the
present invention be administered at any dose for which
chemotherapeutic effects are observed.
In vivo Animal Studies
[0199] A human tumor xenograft model in which OVCAR-5 tumor cells
are grafted into nude mice can be constructed using standard
techniques and materials. The mice are divided into two groups. One
group is treated intraperitoneally with a thiophosphoramidate
oligonucleotides of the invention. The other group is treated with
a control comprising a mixture of phosphate buffer solution (PBS)
and an oligonucleotide complementary with telomerase RNA but has at
least a one base mismatch with the sequence of telomerase RNA. The
average tumor mass for mice in each group is determined
periodically following the xenograft using standard methods and
materials.
[0200] In the group treated with a thiophosphoramidate
oligonucleotide of the invention, the average tumor mass is
expected to increase following the initial treatment for a period
of time, after which time the tumor mass is expected to stabilize
and then begin to decline. Tumor masses in the control group are
expected to increase throughout the study. Thus, the
thiophosphoramidate oligonucleotides of the invention are expected
to lessen dramatically the rate of tumor growth and ultimately
induce reduction in tumor size and elimination of the tumor.
[0201] Thus, the present invention provides novel
thiophosphoramidate oligonucleotides and methods for inhibiting
telomerase activity and treating disease states in which telomerase
activity has deleterious effects, especially cancer. The
thiophosphoramidate oligonucleotides of the invention provide a
highly selective and effective treatment for malignant cells that
require telomerase activity to remain immortal; yet, without
affecting non-malignant cells.
[0202] All printed patents and publications referred to in this
application are hereby incorporated herein in their entirety by
this reference.
[0203] While the preferred embodiment of the invention has been
illustrated and described, it will be appreciated that various
changes can be made therein without departing from the spirit and
scope of the invention.
Sequence CWU 1
1
9 1 11 DNA Artificial Sequence Synthetic oligonucleotide with
potential inhibition activity 1 gttagggtta g 11 2 13 DNA Artificial
Sequence Synthetic oligonucleotide with potential inhibition
activity 2 tagggttaga caa 13 3 11 DNA Artificial Sequence Synthetic
oligonucleotide with potential inhibition activity 3 gttgagtgta g
11 4 13 DNA Artificial Sequence Synthetic oligonucleotide with
potential inhibition activity 4 taggtgtaag caa 13 5 22 DNA
Artificial Sequence Synthetic oligonucleotide with potential
inhibition activity 5 gtagacctgt taccagaggg ag 22 6 26 DNA
Artificial Sequence Synthetic oligonucleotide with potential
inhibition activity 6 ctaaccctaa ctggtaacag gtctac 26 7 18 DNA
Artificial Sequence Synthetic oligonucleotide with potential
inhibition activity 7 ttagggttag ggttaggg 18 8 13 DNA Artificial
Sequence Synthetic oligonucleotide with potential inhibition
activity 8 cagttagggt tag 13 9 11 DNA Artificial Sequence Synthetic
oligonucleotide with potential inhibition activity 9 gattgggatt g
11
* * * * *