Note: Descriptions are shown in the official language in which they were submitted.
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HIGH SPECIFICITY HAIRPIN ANTISENSE OLIGONUCLEOTIDES
This invention relates to antisense oligonucleotides and their therapeutic
use.
Background of the Invention
Antisense oligonucleotides are therapeutic agents. Conventional antisense
oligonucleotides are linear oligonucleotide sequences that are designed to
bind to
target sequences in messenger RNAs and thereby inhibit the translation of the
messenger RNA into its encoded protein, or initiate a chain of events that
causes the
degradation of the messenger RNA with the effect that its encoded protein
cannot be
synthesized. The therapeutic use of antisense oligonucleotides may be hampered
by
poor specificity. For some applications of antisense oligonucleotides, the
sequence of
each messenger RNA is widely different from other messenger RNAs, and
specificity
is not a limitation. However, that is not always true. In some diseases the
translation
of a "wild-type" messenger RNA does not cause a pathogenic condition, but if a
particular nucleotide substitution is present in that messenger RNA, then its
expression does cause a pathogenic condition. It is desirable to be able to
use an
antisense oligonucleotide to inhibit the expression of such an RNA. Typically,
the
mutant messenger RNA is associated with cells that are cancerous. For example,
the
human ras gene becomes cancer-inducing by the acquisition of a single
nucleotide
point mutation within its coding sequence (Monia et al., 1992). In this and
similar
situations, it is important that the antisense oligonucleotide bind to the
cancer-causing
variant of the ras messenger RNA, but that it not bind to the normal ras
messenger
RNA that is present in healthy cells.
Relatively long oligonucleotides are used as antisense agents in order that
they form strong hybrids and select against unrelated messenger RNAs. These
agents
do not discriminate in practice against single nucleotide differences.
Shortening the
length of the antisense oligonucleotide to improve its specificity for the
mutant target
sequence as compared to the almost identical wild-type sequence is not very
useful,
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because this weakens the hybrids that it forms, and as a result it is not able
to inhibit
the expression of mutant messengers RNA.
The present invention markedly improves the specificity of antisense
oligonucleotides and antisense therapy.
Summary of the Invention
This invention includes modified antisense oligonucleotides having
increased specificity. The antisense oligonucleotides of this invention, when
not
bound to target, assume a structured hairpin configuration comprising a single-
stranded loop and a double-stranded stem. The loop is complementary to the
target.
Interaction of the loop to the target results in the formation of a loop-
target hybrid,
which causes the stem to unwind, or dissociate. Antisense oligonucleotides
according
to this invention are able to discriminate between an intended target
messenger RNA
and another messenger RNA differing from the target by a single nucleotide
substitution.
Antisense oligonucleotides according to this invention have an internal
sequence flanked by a pair of sequences that are complementary to one another
and
hybridize to one another under conditions of use in the absence of target
messenger
RNA. We refer to the three sequences as the 5' arm, the loop, and the 3' arm.
Hybridization to the arms to one another creates a double-stranded region, or
stem.
Hybridization of the loop sequence to its perfectly complementary target
sequence
causes the stem to dissociate, or unwind. This hairpin structure, which is not
possessed by conventional linear antisense oligonucleotides, markedly improves
the
ability to discriminate between two messenger RNAs that differ by a single
nucleotide.
This invention also includes therapeutic use of these hairpin antisense
oligonucleotide, which may be administered in the conventional fashion already
known for linear antisense oligonucleotides, for example, by encapulating the
agents
in liposomes that fuse with all membranes and thereby deliver there contents
into
cells. Antibodies anchored on liposomes can target the liposomes to cancerous
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tumors. Administration is in a therapeutically effective amount by which we
mean an
amount that is capable of producing a medically desirable result in a treated
mammal,
for example, a human patient.
Description of the Preferred Embodiments
The use of extremely specific hairpin antisense oligonucleotides that
selectively inhibit the expression of a pathogenic gene, without inhibiting
the
expression of a related gene that differs from it by only a single nucleotide
substitution, is based on the finding that the presence of the hairpin stem
enhances the
specificity of the probe sequence located in the loop of the hairpin
structure. Hairpin
antisense oligonucleotides are simple to design. The arm sequences of hairpin
antisense oligonucleotides can be chosen independently of the identity of the
target
sequence. Only the loop portion of the hairpin antisense oligonucleotide needs
to be
complementary to the target. The loop sequences in hairpin antisense
oligonucleotides are sufficiently long to be specific to a chosen sequence and
relatively long as compared to the arms such that hybridization of the loop
sequence
alone drives the opening of the hairpin stem. The length of the loop sequence
in a
hairpin antisense deoxyriboligonucleotide according to this invention ranges
from 7 to
30 nucleotides, preferably 10 to 25 nucleotides and more preferably 15-25
nucleotides, whereas the length of the arm sequence ranges from 3 to 8
nucleotides,
with the loop sequence always being longer than the stem sequence. The
specificity
of hairpin antisense oligonucleotides increases as the length (or GC content)
of the
arm sequences is increased. Whether a particular hairpin antisense
oligonucleotide
actually exhibits the desired level of specificity can be tested in vitro by
labeling the
hairpin antisense oligonucleotide with terminal interactive labels according
to the
methods of Tyagi and Kramer (1996) and then detecting hybridization by
observing
the increase in fluorescence intensity. A hairpin antisense oligonucleotide
will bind to
a perfectly complementary target sequence in a messenger RNA, that is, a
target
perfectly complementary to the loop, but will not bind to a sequence in a
messenger
RNA that differs from the target sequence by a single nucleotide substitution,
and,
thus, is not perfectly complementary to the loop.
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Another useful aspect of hairpin antisense oligonucleotides is that because
they are in the form of a hairpin that possesses a double-stranded stem, they
are
naturally more resistant to cellular nucleases than conventional linear
antisense
oligonucleotides.
Hairpin antisense oligonucleotides of the present invention can contain
deoxyribonucleotides, ribonucleotides, peptide nucleic acids (PNA), other
modified
nucleotides, or combinations of these. Modified nucleotides may include, for
example, 2'-O-methylribonucleotides or nitropyrole-based nucleotides. Modified
internucleotide linkages may also be included, for example phosphorothioates.
The
advantage of using such modifications for a particular application will be
apparent to
persons familiar with the art. In particular, hairpin antisense
oligonucleotides
constructed from modified nucleotides may form stronger hybrids than if the
hairpin
primers were constructed from deoxyribonucleotides, thus enabling structured
target
sequences (such as those that occur in messenger RNAs) to be more easily
accessed.
In addition, hairpin antisense oligonucleotides constructed from modified
nucleotides,
or that contain modified internucleotide linkages, will resist degradation by
cellular
nucleases, rendering them more effective as therapeutic agents.
Hairpin antisense oligonucleotides according to this invention may have an
arm that is at least partially complementary to the intended target. However,
it must
open when tested against a target consisting of only the perfect complement of
the
loop.
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Example
We have designed a highly specific hairpin antisense oligonucleotide that
binds to a mutant carcinogenic form of ras messenger RNA, but does not bind to
the
wild-type form of ras messenger RNA. These two RNAs differ from one another by
a single nucleotide substitution within codon 12 (Monia et al., 1992). This
embodiment is made of deoxyribonucleotides. The sequence of the hairpin
antisense
oligonucleotide is 5'-CGCTGGCCCGCGGCAGCCACACCCCAGCG-3', where
underlines identify the arm sequences that hybridize to each other, the 5' arm
being
the sequence CGCTGG. In the absence of target strands that are complementary
to
the single-stranded loop, the two arms hybridize to one another to form a
stem. If the
stem sequences had not been added to the loop sequence in this antisense
oligonucleotide, it would not have sufficient capacity to discriminate between
wild-type ras messenger RNA and mutant ras messenger RNA (Monia et al., 1992).
However, the hairpin antisense oligonucleotide is much more specific for its
intended
target sequence because the sequence in the loop must initiate the binding of
the
oligonucleotide to the target sequence, and this interaction is much more
specific than
the binding of a conventional linear antisense oligonucleotide to the same
target
sequence because the interactive sequence in the loop is embedded within a
hairpin
stem. By only binding to mutant ras RNA, hairpin antisense oligonucleotides
selectively inhibit the growth of cells that express the mutant messenger RNA
(cancer
cells), and do not inhibit the growth of cells that express the wild-type
messenger
RNA (healthy cells).
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References
Monia, B. P., Johnston, J. F., Ecker, D. J., Zounes, M. A., Lima, W. F., and
Freier, S. M. ( 1992) Selective inhibition of mutant Ha-ras mRNA expression by
antisense oligonucleotides. J. Biol. Chem. 267, 19954-19962.
Tyagi, S., and Kramer, F. R. (1996) Molecular beacons: probes that fluoresce
upon hybridization. Nat. Biotechnol. 14, 303-308.
Tyagi, S., Bratu, D. P., and Kramer, F. R. (1998) Multicolor molecular
beacons for allele discrimination. Nat. Biotechnol. 16, 49-53.
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