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CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
NUCLEIC ACID BASED MODULATORS OF GENE EXPRESSION
Background of the Invention
This invention relates to reagents useful as inhibitors of gene expression
relating to
diseases such as cancers, diabetes, obesity, Alzheimer's disease, cardiac
diseases, age-
s related diseases, and/or hepatitis B infections and related conditions.
Summary,of the Invention
The invention features novel nucleic acid-based techniques [e.g., enzymatic
nucleic
acid molecules (ribozymes), antisense nucleic acids, 2-5A antisense chimeras,
triplex
DNA, antisense nucleic acids containing RNA cleaving chemical groups (fox
example,
Cook et aL, U.S. Patent 5,359,051)] and methods for their use to modulate the
expression
of molecular targets impacting the development and progression of cancers,
diabetes,
obesity, Alzheimer's disease, cardiac diseases, age-related diseases, and/or
hepatitis B
infections and related conditions
In a preferred embodiment, the invention features novel nucleic acid-based
techniques [e.g., enzymatic nucleic acid molecules (ribozymes), antisense
nucleic acids, 2-
SA antisense chimeras, triplex DNA, antisense nucleic acids containing RNA
cleaving
chemical groups (for exaple, Cook et al., U.S. Patent 5,359,051)] and methods
for their use
for inhibiting the expression of disease related genes, e.g., Protein-Tyrosine-
Phosphatase-
1b (PTP-1B, Genbank accession No. NM~002827), Methionine Aminopeptidase (MetAP-
2, Genbank accession No. U29607), beta-Secretase (BACE, Genbank accession No.
AF190725), Presenilin-1 (ps-1, Genbank accession No. L76517), Presenilin-2 (ps-
2,
Genbank accession No. L43964), Human Epidermal Growth Factor Receptor-2
(HER2/c-
erb2/neu, Genbank accession No. X03363), Phospholamban (PLN, Genbank accession
No.
NM 002667), Telomerase (TERT, Genbank accession No. NM_003219) and Hepatitis B
virus genes (HBV, Genbank accession No. AF100308.1). Such ribozymes can be
used in
a method for treatment of diseases caused by the expression of these genes in
man and
other animals, including other primates.
Thus, in an additional preferred embodiment, the invention features novel
nucleic
acid-based techniques such as enzymatic nucleic acid molecules and antisense
molecules
and methods for their use to down regulate or inhibit the expression of genes
encoding
Protein-Tyrosine-Phosphatase-lb (PTP-1B), Methionine Aminopeptidase (MetAP-2),
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beta-Secretase (BACE), Presenilin-1 (ps-1), Presenilin-2 (ps-2), Human
Epidermal Growth
Factor Receptor-2 (HER2/c-erb2/neu), Phospholamban (PLN), Telomerase (hTERT)
PKC
alpha. and Hepatitis B (HBV) proteins. In particular, applicant describes the
selection and
function of nucleic acid molecules capable of cleaving RNAs encoded by these
genes and
their use to reduce levels of PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN,
TERT,
and/or HBV proteins in various tissues to treat the diseases discussed herein.
Such nucleic
acid molecules are also useful for diagnostic uses.
In a preferred embodiment, the invention features the use of one or more of
the
nucleic acid-based techniques independently or in combination to inhibit the
expression of
the genes encoding PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or
HBV. Specifically, the invention features the use of nucleic acid-based
techniques to
specifically inhibit the expression of PTP-1B, MetAP-2, BACE, ps-1, ps-2,
HER2, PLN,
TERT, PKC alpha, and/or HBV genes.
In yet another preferred embodiment, the invention features the use of an
enzymatic
nucleic acid molecule, preferably in the hammerhead, NCH (Inozyme), G-cleaver,
amberzyme, zinzyme, and/or DNAzyme motif, to inhibit the expression of PTP-1B,
MetAP-2, BALE, ps-1, ps-2, HER2, PLN, TERT, PKC alpha and/or HBV RNA.
Applicant indicates that these nucleic acid molecules are able to inhibit
expression of
PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, PKC alpha, and/or HBV
genes. Those of ordinary skill in the art, will find that it is clear from the
examples
described that other nucleic acid molecules that inhibit target PTP-1B, MetAP-
2, BACE,
ps-1, ps-2, HER2, PLN, TERT, and/or HBV encoding mRNAs may be readily designed
and are within the scope of the invention.
By "inhibit" it is meant that the activity of target genes or level of mRNAs
or
equivalent RNAs encoding target genes is reduced below that observed in the
absence of
the nucleic acid molecules of the instant invention (e.g., enzymatic nucleic
acid
molecules), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA,
antisense
nucleic acids containing RNA cleaving chemical groups). In one embodiment,
inhibition
with an enzymatic nucleic acid molecule preferably is below that level
observed in the
presence of an enzymatically attenuated nucleic acid molecule that is able to
bind to the
same site on the mRNA, but is unable to cleave that RNA. In another
embodiment,
inhibition with nucleic acid molecules, including enzymatic nucleic acid and
antisense
CA 02403243 2002-02-21
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molecules, is preferably greater than that observed in the presence of, for
example, an
oligonucleotide with scrambled sequence or with mismatches. In another
embodiment,
inhibition of target genes with the nucleic acid molecule of the instant
invention is greater
than in the presence of the nucleic acid molecule than in its absence.
According to the
invention, the activity of telomerase enzyme or the level of RNA encoding one
or more
portein subunits of the telomerase enzyme is inhibited if it is at least 10%
less, 20% less,
50% less, 75% less or even not active or present at all, in the presence of a
nucleic acid of
the invention relative to the level in the absence of such a nucleic acid.
By "enzymatic nucleic acid molecule" it is meant a nucleic acid molecule which
has
complementarity in a substrate binding region to a specified gene target, and
also has an
enzymatic activity which is active to specifically cleave target RNA. That is,
the
enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and
thereby
inactivate a target RNA molecule. These complementary regions allow sufficient
hybridization of the enzymatic nucleic acid molecule to the target RNA and
thus permit
cleavage. One hundred percent complementarity is preferred, but
complementarity as low
as 50-75% may also be useful in this invention. The nucleic acids may be
modified at the
base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used
interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA,
catalytic
DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic
oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease,
endonuclease,
minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies
describe
nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic
acid
molecules described in the instant application are not meant to be limiting
and those
skilled in the art will recognize that all that is important in an enzymatic
nucleic acid
molecule of this invention is that it have a specific substrate binding site
which is
complementary to one or more of the target nucleic acid regions, and that it
have
nucleotide sequences within or surrounding that substrate binding site which
impart a
nucleic acid cleaving activity to the molecule (Cech et al., U.S. Patent No.
4,987,071;
Cech et al., 1988, JAMA 260:20 3030-4).
By "nucleic acid molecule" as used herein is meant a molecule having
nucleotides.
The nucleic acid can be single, double, or multiple stranded and may comprise
modified or
unmodified nucleotides or non-nucleotides or various mixtures and combinations
thereof.
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An example of a nucleic acid molecule according to the invention is a gene
which encodes
for a macromolecule such as a protein.
By "enzymatic portion" or "catalytic domain" is meant that portion/region of
the
enzymatic nucleic acid molecule essential for cleavage of a nucleic acid
substrate (for
example see Figures 1-5).
By "substrate binding arm" or "substrate binding domain" is meant that
portion/region of a ribozyme which is complementary to (i.e., able to base-
pair with) a
portion of its substrate. Generally, such complementarity is 100%, but can be
less if
desired. For example, as few as 10 bases out of 14 may be base-paired. Such
arms are
shown generally in Figures 1-5. That is, these arms contain sequences within a
ribozyme
which are intended to bring ribozyme and target RNA together through
complementary
base-pairing interactions. The ribozyme of the invention may have binding arms
that are
contiguous or non-contiguous and may be of varying lengths. The length of the
binding
arms) are preferably greater than or equal to four nucleotides and of
sufficient length to
stably interact with the target RNA; specifically 12-100 nucleotides; more
specifically 14-
24 nucleotides long. If two binding arms are chosen, the design is such that
the length of
the binding arms are symmetrical (i.e., each of the binding arms is of the
same length; e.g.,
five and five nucleotides, six and six nucleotides or seven and seven
nucleotides long) or
asymmetrical (i.e., the binding arms are of different length; e.g., six and
three nucleotides;
three and six nucleotides long; four and five nucleotides long; four and six
nucleotides
long; four and seven nucleotides long; and the like). Binding arms can be
complementary
to the specified substrate, to a portion of the indicated substrate, to the
indicated substrate
sequence and additional adjacent sequence, or a portion of the indicated
sequence and
additional adjacent sequence.
By "NCH" or "Inozyme" motif is meant, an enzymatic nucleic acid molecule
comprising a motif as described in Ludwig et al., USSN No. 09/406,643, filed
September
27, 1999, entitled "COMPOSITIONS HAVING RNA CLEAVING ACTIVITY", and
International PCT publication Nos. WO 98/58058 and WO 98/58057, all
incorporated by
reference herein in their entirety, including the drawings.
By "G-cleaver" motif is meant, an enzymatic nucleic acid molecule comprising a
motif as described in Eckstein et al., International PCT publication No. WO
99/16871,
incorporated by reference herein in its entirety, including the drawings.
CA 02403243 2002-02-21
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By "zinzyme" motif is meant, a class II enzymatic nucleic acid molecule
comprising
a motif as described herein and in Beigelman et al., International PCT
publication No. WO
99/55857, incorporated by reference herein in its entirety, including the
drawings.
By "amberzyme" motif is meant, a class I enzymatic nucleic acid molecule
comprising a motif as described herein and in Beigelman et al., International
PCT
publication No. WO 99/55857, incorporated by reference herein in its entirety,
including
the drawings.
By 'DNAzyme' is meant, an enzymatic nucleic acid molecule lacking a
ribonucleotide (2'-OH) group. In particular embodiments, the enzymatic nucleic
acid
molecule may have an attached linkers) or other attached or associated groups,
moieties,
or chains containing one or more nucleotides with 2'-OH groups. A DNAzyme can
be
synthesized chemically or can be expressed by means of a single stranded DNA
vector or
equivalent thereof.
By "sufficient length" is meant an oligonucleotide of greater than or equal to
3
nucleotides that is of a length great enough to provide the intended function
under the
expected condition. For example, for binding arms of enzymatic nucleic acid
"sufficient
length" means that the binding arm sequence is long enough to provide stable
binding to a
target site under the expected binding conditions. Preferably, the binding
arms are not so
long as to prevent useful turnover.
By "stably interact" is meant, interaction of the oligonucleotides with target
nucleic
acid (e.g., by forming hydrogen bonds with complementary nucleotides in the
target under
physiological conditions).
By "equivalent" RNA to PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT,
and/or HBV is meant to include those naturally occurring RNA molecules having
homology (partial or complete) to PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2,
PLN,
TERT, and/or HBV proteins or encoding for proteins with similar function as
PTP-1B,
MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV in various organisms,
including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and
other
microorganisms and parasites. The equivalent RNA sequence also includes in
addition to
the coding region, regions such as 5'-untranslated region, 3'-untranslated
region, introns,
intron-exon junction and the like in HBV.
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6
By "homology" is meant the nucleotide sequence of two or more nucleic acid
molecules is partially or completely identical.
By "antisense nucleic acid", it is meant a non-enzymatic nucleic acid molecule
that
binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic
acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the
activity of the target
RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et
al., US
patent No. 5,849,902). Typically, antisense molecules will be complementary to
a target
sequence along a single contiguous sequence of the antisense molecule.
However, in
certain embodiments, an antisense molecule may bind to substrate such that the
substrate
molecule forms a loop, and/or an antisense molecule may bind such that the
antisense
molecule forms a loop. Thus, the antisense molecule may be complementary to
two (or
even more) non-contiguous substrate sequences or two (or even more) non-
contiguous
sequence portions of an antisense molecule may be complementary to a target
sequence or
both. For a review of current antisense strategies, see Schmajuk et al., 1999,
J. Biol.
Chem., 274, 21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et
al., 1997,
Antisense N. A. Drug Dev., 7, 151, Crooke, 1998, Biotech. Genet. Eng. Rev.,
15, 121-157,
Crooke, 1997, Ad. Pharmacol., 40, 1-49. In addition, antisense DNA can be used
to target
RNA by means of DNA-RNA interactions, thereby activating RNase H, which
digests the
target RNA in the duplex. Antisense DNA can be synthesized chemically or can
be
expressed via the use of a single stranded DNA expression vector or the
equivalent
thereof.
By "2-SA antisense chimera" it is meant, an antisense oligonucleotide
containing a
S'-phosphorylated 2'-5'-linked adenylate residue. These chimeras bind to
target RNA in a
sequence-specific manner and activate a cellular 2-SA-dependent ribonuclease
which, in
turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA
90, 1300).
By "triplex DNA" it is meant an oligonucleotide that can bind to a double-
stranded
DNA in a sequence-specific manner to form a triple-strand helix. Formation of
such triple
helix structure has been shown to inhibit transcription of the targeted gene
(Duval-
Valentin et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 504).
By "gene" it is meant a nucleic acid that encodes a RNA.
CA 02403243 2002-02-21
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By "complementarity" is meant that a nucleic acid can form hydrogen bonds)
with
another RNA sequence by either traditional Watson-Crick or other non-
traditional types.
In reference to the nucleic molecules of the present invention, the binding
free energy for a
nucleic acid molecule with its target or complementary sequence is sufficient
to allow the
relevant function of the nucleic acid to proceed, e.g., ribozyme cleavage,
antisense or triple
helix inhibition. Determination of binding free energies for nucleic acid
molecules is well
known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII
pp.123-133;
Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al.,
1987, J. Am.
Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage
of
contiguous residues in a nucleic acid molecule which can form hydrogen bonds
(e.g.,
Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7,
8, 9, 10 out
of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly
complementary" means that all the contiguous residues of a nucleic acid
sequence will
hydrogen bond with the same number of contiguous residues in a second nucleic
acid
sequence.
At least seven basic varieties of naturally-occurnng enzymatic RNAs are known
presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in
traps (and
thus can cleave other RNA molecules) under physiological conditions. Table I
summarizes some of the characteristics of these ribozymes. In general,
enzymatic nucleic
acids act by first binding to a target RNA. Such binding occurs through the
target binding
portion of a enzymatic nucleic acid which is held in close proximity to an
enzymatic
portion of the molecule that acts to cleave the target RNA. Thus, the
enzymatic nucleic
acid first recognizes and then binds a target RNA through complementary base-
pairing,
and once bound to the correct site, acts enzymatically to cut the target RNA.
Strategic
cleavage of such a target RNA will destroy its ability to direct synthesis of
an encoded
protein. After an enzymatic nucleic acid has bound and cleaved its RNA target,
it is
released from that RNA to search for another target and can repeatedly bind
and cleave
new targets. Thus, a single ribozyme molecule is able to cleave many molecules
of target
RNA. In addition, the ribozyme is a highly specific inhibitor of gene
expression, with the
specificity of inhibition depending not only on the base-pairing mechanism of
binding to
the target RNA, but also on the mechanism of target RNA cleavage. Single
mismatches,
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WO 01/16312 PCT/US00/23998
or base-substitutions, near the site of cleavage can completely eliminate
catalytic activity
of a ribozyme.
The enzymatic nucleic acid molecule that cleave the specified sites in PTP-1B,
MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV-specific RNAs represent
a
novel therapeutic approach to treat a variety of pathologic indications,
including, HBV
infection, hepatitis, hepatocellular carcinoma, tumorigenesis, cirrhosis,
liver failure,
cancers including breast, ovarian, prostate, and esophogeal cancer,
tumorigenesis,
retinopathy, arthritis, psoriasis, female reproduction, restinosis, certain
infectious diseases,
transplant rejection and autoimmune disease such as multiple sclerosis, lupus,
and AIDS,
age related diseases such as macular degeneration and skin ulceration,
Alzheimer's
disease, dementia, diabetes, obesity and any other condition related to the
level of PTP-1B,
MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV in a cell or tissue.
In one of the preferred embodiments of the inventions described herein, the
enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif,
but may also
be formed in the motif of a hepatitis delta virus, group I intron, group II
intron or RNase P
RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes,
NCH cleaving motifs, or G-cleavers. Examples of such hammerhead motifs are
described
by Dreyfus, supra, Rossi et al., 1992, AIDS Research and Human Retroviruses 8,
183.
Examples of hairpin motifs are described by Hampel et al., EP0360257, Hampel
and Tritz,
1989 Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53, Haseloff and
Gerlach,
1989, Gene, 82, 43, Hampel et al., 1990 Nucleic Acids Res. 18, 299; and
Chowrira &
McSwiggen, US. Patent No. 5,631,359. The hepatitis delta virus motif is
described by
Perrotta and Been, 1992 Biochemistry 31, 16. The RNase P motif is described by
Guerner-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990, Science
249, 783;
and Li and Altman, 1996, Nucleic Acids Res. 24, 835. The Neurospora VS RNA
ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-
696;
Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins
and Olive,
1993 Biochemistry 32, 2795-2799; and Guo and Collins, 1995, EMBO. J. 14, 363).
Group
II introns are described by Griffin et al., 1995, Chem. Biol. 2, 761; Michels
and Pyle, 1995,
Biochemistry 34, 2965; and Pyle et al., International PCT Publication No. WO
96/22689.
The Group I intron is described by Cech et al., U.S. Patent 4,987,071.
DNAzymes are
described by Usman et al., International PCT Publication No. WO 95/11304;
Chartrand et
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WO 01/16312 PCT/US00/23998
al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; and Santoro
et al., 1997,
PNAS 94, 4262. NCH cleaving motifs are described in Ludwig & Sproat,
International
PCT Publication No. WO 98/58058; and G-cleavers are described in Kore et al.,
1998,
Nucleic Acids Research 26, 4116-4120 and Eckstein et al., International PCT
Publication
No. WO 99/16871. Additional motifs include the Aptazyme (Breaker et al., WO
98/43993), Amberzyme (Class I motif; Figure 3; Beigelman et al., International
PCT
publication No. WO 99/55857) and Zinzyme (Beigelinan et al., International PCT
publication No. WO 99/55857), all these references are incorporated by
reference herein in
their totalities, including drawings and can also be used in the present
invention. These
specific motifs are not limiting in the invention and those skilled in the art
will recognize
that all that is important in an enzymatic nucleic acid molecule of this
invention is that it
has a specific substrate binding site which is complementary to one or more of
the target
gene RNA regions, and that it have nucleotide sequences within or surrounding
that
substrate binding site which impart an RNA cleaving activity to the molecule
(Cech et al.,
U.S. Patent No. 4,987,071).
In preferred embodiments of the present invention, a nucleic acid molecule,
e.g., an
antisense molecule, a triplex DNA, or a ribozyme, is 13 to 100 nucleotides in
length, e.g.,
in specific embodiments 35, 36, 37, or 38 nucleotides in length (e.g., for
particular
ribozymes or antisense). In particular embodiments, the nucleic acid molecule
is 15-100,
17-100, 20-100, 21-100, 23-100, 25-100, 27-100, 30-100, 32-100, 35-100, 40-
100, 50-100,
60-100, 70-100, or 80-100 nucleotides in length. Instead of 100 nucleotides
being the
upper limit on the length ranges specified above, the upper limit of the
length range can be,
for example, 30, 40, 50, 60, 70, or 80 nucleotides. Thus, for any of the
length ranges, the
length range for particular embodiments has lower limit as specified, with an
upper limit
as specified which is greater than the lower limit. For example, in a
particular
embodiment, the length range can be 35-50 nucleotides in length. All such
ranges are
expressly included. Also in particular embodiments, a nucleic acid molecule
can have a
length which is any of the lengths specified above, for example, 21
nucleotides in length.
In a preferred embodiment, the invention provides a method for producing a
class of
nucleic acid based gene inhibiting agents which exhibit a high degree of
specificity for the
RNA of a desired target. For example, the enzymatic nucleic acid molecule is
preferably
targeted to a highly conserved sequence region of target RNAs encoding PTP-1B,
MetAP-
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2, BACE, ps-l, ps-2, HER2, PLN, TERT, and/or HBV proteins (specifically PTP-
1B,
MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV RNA) such that specific
treatment of a disease or condition can be provided with either one or several
nucleic acid
molecules of the invention. Such nucleic acid molecules can be delivered
exogenously to
5 specific tissue or cellular targets as required. Alternatively, the nucleic
acid molecules
(e.g., ribozymes and antisense) can be expressed from DNA and/or RNA vectors
that are
delivered to specific cells.
As used in herein "cell" is used in its usual biological sense, and does not
refer to an
entire multicellular organism, e.g., specifically does not refer to a human.
The cell may be
10 present in an organism which may be a human but is preferably a non-human
multicellular
organism, e.g., birds, plants and mammals such as cows, sheep, apes, monkeys,
swine,
dogs, and cats. The cell may be prokaryotic (e.g., bacterial cell) or
eukaryotic (e.g.,
mammalian or plant cell).
By "PTP-1B, MetAP-2, BACE, ps-l, ps-2, HER2, PLN, TERT, and/or HBV
proteins" is meant, a protein or a mutant protein derivative thereof,
comprising sequence
expressed and/or encoded by PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN,
TERT,
genes and/or the HBV genome respectively.
By "highly conserved sequence region" is meant a nucleotide sequence of one or
more regions in a target gene does not vary significantly from one generation
to the other
or from one biological system to the other.
The enzymatic nucleic acid-based inhibitors of PTP-1B, MetAP-2, BACE, ps-l, ps-
2, HER2, PLN, TERT, and/or HBV expression are useful for the prevention of the
diseases
and conditions including HBV infection, hepatitis, hepatocellular carcinoma,
tumorigenesis, cirrhosis, liver failure, cancers including breast, ovarian,
prostate, and
esophogeal cancer, tumorigenesis, retinopathy, arthritis, psoriasis, female
reproduction,
restinosis, certain infectious diseases, transplant rejection and autoimmune
disease such as
multiple sclerosis, lupus, and AIDS, age related diseases such as macular
degeneration and
skin ulceration, Alzheimer's disease, dementia, diabetes, obesity and any
other condition
related to the level of PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT,
and/or
HBV in a cell or tissue. and any other diseases or conditions that are related
to the levels
of PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV in a cell or
tissue.
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11
By "related" is meant that the reduction of PTP-1B, MetAP-2, BACE, ps-1, ps-2,
HER2, PLN, TERT, and/or HBV expression (specifically PTP-1B, MetAP-2, BACE, ps-
1,
ps-2, HER2, PLN, TERT, and/or HBV genes) RNA levels and thus reduction in the
level
of the respective protein will relieve, to some extent, the symptoms of the
disease or
condition.
The nucleic acid-based inhibitors of the invention are added directly, or can
be
complexed with cationic lipids, packaged within liposomes, or otherwise
delivered to
target cells or tissues. The nucleic acid or nucleic acid complexes can be
locally
administered to relevant tissues ex vivo, or in vivo through injection,
infusion pump or
stmt, with or without their incorporation in biopolymers. In preferred
embodiments, the
enzymatic nucleic acid inhibitors comprise sequences, which are complementary
to the
substrate sequences in Tables 3-31, 33, 34, 36-43, 56, 58, 59, 62, 63.
Examples of such
enzymatic nucleic acid molecules also are shown in Tables 3-29, 31, 33, 34, 37-
43, 56, 58,
59, 62, 63. Examples of such enzymatic nucleic acid molecules consist
essentially of
sequences defined in these tables.
1n yet another embodiment, the invention features antisense nucleic acid
molecules
including sequences complementary to the substrate sequences shown in Tables 3-
31, 33,
34, 36, 37-43, 56, 58, 59, 62, 63. Such nucleic acid molecules can include
sequences as
shown for the binding arms of the enzymatic nucleic acid molecules in Tables 3-
29, 31,
33, 34, 37-43, 56, 58, 59, 62, 63. Similarly, triplex molecules can be
provided targeted to
the corresponding DNA target regions, and containing the DNA equivalent of a
target
sequence or a sequence complementary to the specified target (substrate)
sequence.
Typically, antisense molecules will be complementary to a target sequence
along a single
contiguous sequence of the antisense molecule. However, in certain
embodiments, an
antisense molecule may bind to substrate such that the substrate molecule
forms a loop,
and/or an antisense molecule may bind such that the antisense molecule forms a
loop.
Thus, the antisense molecule may be complementary to two (or even more) non-
contiguous substrate sequences or two (or even more) non-contiguous sequence
portions
of an antisense molecule may be complementary to a target sequence or both.
In another aspect, the invention provides mammalian cells containing one or
more
nucleic acid molecules and/or expression vectors of this invention. The one or
more
nucleic acid molecules may independently be targeted to the same or different
sites.
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12
By "consists essentially off' is meant that the active nucleic acid molecule
of the
invention, for example, an enzymatic nucleic acid molecule, contains an
enzymatic center
or core equivalent to those in the examples, and binding arms able to bind
mRNA such
that cleavage at the target site occurs. Other sequences may be present which
do not
interfere with such cleavage. Thus, a core region may, for example, include
one or more
loop or stem-loop structures, which do not prevent enzymatic activity. "X" in
the
sequences in Tables 3, 4, 9,10,13,14,18,19, 24, 25, 33, 34, 37, 38, 63 can be
such a
loop. A core sequence for a hammerhead ribozyme can be CUGAUGAG X CGAA where
X=GCCGUUAGGC or other stem II region as specifically or generally known in the
art.
In another aspect of the invention, ribozymes or antisense molecules that
interact
with target RNA molecules and inhibit PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2,
PLN,
TERT, and/or HBV (specifically PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN,
TERT, and/or HBV RNA) activity are expressed from transcription units inserted
into
DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or
viral
vectors. Ribozyme or antisense expressing viral vectors could be constructed
based on,
but not limited to, adeno-associated virus, retrovirus, adenovirus, or
alphavirus.
Preferably, the recombinant vectors capable of expressing the ribozymes or
antisense are
delivered as described above, and persist in target cells. Alternatively,
viral vectors may
be used that provide for transient expression of ribozymes or antisense. Such
vectors
might be repeatedly administered as necessary. Once expressed, the ribozymes
or
antisense bind to the target RNA and inhibit its function or expression.
Delivery of
ribozyme or antisense expressing vectors could be systemic, such as by
intravenous or
intramuscular administration, by administration to target cells ex-planted
from the patient
followed by reintroduction into the patient, or by any other means that would
allow for
introduction into the desired target cell. Antisense DNA can be expressed via
the use of a
single stranded DNA intracellular expression vector.
By RNA is meant a molecule comprising at least one ribonucleotide residue. By
"ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2'
position of a (3-D-
ribo-furanose moiety.
By "vectors" is meant any nucleic acid- and/or viral-based technique used to
deliver
a desired nucleic acid.
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13
By "patient" is meant an organism, which is a donor or recipient of explanted
cells or
the cells themselves. "Patient" also refers to an organism to which the
nucleic acid
molecules of the invention can be administered. Preferably, a patient is a
mammal or
mammalian cells. More preferably, a patient is a human or human cells.
The nucleic acid molecules of the instant invention, individually, or in
combination
or in conjunction with other drugs, can be used to treat diseases or
conditions discussed
above. For example, to treat a disease or condition associated with PTP-1B,
MetAP-2,
BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV, the patient may be treated, or
other
appropriate cells may be treated, as is evident to those skilled in the art,
individually or in
combination with one or more drugs under conditions suitable for the
treatment.
In a further embodiment, the described molecules, such as antisense or
ribozymes,
can be used in combination with other known treatments to treat conditions or
diseases
discussed above. For example, the described molecules could be used in
combination with
one or more known therapeutic agents to treat HBV infection, hepatitis,
hepatocellular
carcinoma, tumorigenesis, cirrhosis, liver failure, cancers including breast,
ovarian,
prostate, and esophogeal cancer, tumorigenesis, retinopathy, arthritis,
psoriasis, female
reproduction, restinosis, certain infectious diseases, transplant rejection
and autoimmune
disease such as multiple sclerosis, lupus, and A>DS, age related diseases such
as macular
degeneration and skin ulceration, Alzheimer's disease, dementia, diabetes,
and/or obesity.
In another preferred embodiment, the invention features nucleic acid-based
inhibitors (e.g., enzymatic nucleic acid molecules (ribozymes), antisense
nucleic acids,
triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups)
and
methods for their use to down regulate or inhibit the expression of RNA (e.g.,
PTP-1B,
MetAP-2, BACE, ps-l, ps-2, HER2, PLN, TERT, and/or HBV) capable of progression
and/or maintenance of HBV infection, hepatitis, hepatocellular carcinoma,
tumorigenesis,
cirrhosis, liver failure, cancers including breast, ovarian, prostate, and
esophogeal cancer,
tumorigenesis, retinopathy, arthritis, psoriasis, female reproduction,
restinosis, certain
infectious diseases, transplant rejection and autoimmune disease such as
multiple sclerosis,
lupus, and A)DS, age related diseases such as macular degeneration and skin
ulceration,
Alzheimer's disease, dementia, diabetes, and/or obesity.
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14
In another preferred embodiment, the invention features nucleic acid-based
techniques (e.g., enzymatic nucleic acid molecules (ribozymes), antisense
nucleic acids,
triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups)
and
methods for their use to down regulate or inhibit the expression of PTP-1B,
MetAP-2,
BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV RNA expression.
By "comprising" is meant including, but not limited to, whatever follows the
word
"comprising". Thus, use of the term "comprising" indicates that the listed
elements are
required or mandatory, but that other elements are optional and may or may not
be present.
By "consisting of is meant including, and limited to, whatever follows the
phrase
"consisting of'. Thus, the phrase "consisting of indicates that the listed
elements are
required or mandatory, and that no other elements may be present. By
"consisting
essentially of is meant including any elements listed after the phrase, and
limited to other
elements that do not interfere with or contribute to the activity or action
specified in the
disclosure for the listed elements. Thus, the phrase "consisting essentially
of indicates
that the listed elements are required or mandatory, but that other elements
are optional and
may or may not be present depending upon whether or not they affect the
activity or action
of the listed elements.
Other features and advantages of the invention will be apparent from the
following
description of the preferred embodiments thereof, and from the claims.
Description Of The Preferred Embodiments
The drawings will first briefly be described.
Drawings:
Figure 1 shows the secondary structure model for seven different classes of
enzymatic nucleic acid molecules. Arrow indicates the site of cleavage. -------
-- indicate
the target sequence. Lines interspersed with dots are meant to indicate
tertiary interactions.
- is meant to indicate base-paired interaction. Group I Intron: P1-P9.0
represent various
stem-loop structures (Cech et al., 1994, Nature Struc. Bio., 1, 273). RNase P
(M1RNA):
EGS represents external guide sequence (Forster et al., 1990, Science, 249,
783; Pace et
al., 1990, J. Biol. Chem., 265, 3587). Group II Intron: 5'SS means 5' splice
site; 3'SS
means 3'-splice site; IBS means intron binding site; EBS means exon binding
site (Pyle et
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al., 1994, Biochemistry, 33, 2716). VS RNA: I-VI are meant to indicate six
stem-loop
structures; shaded regions are meant to indicate tertiary interaction
(Collins, International
PCT Publication No. WO 96/19577). HDV Ribozyme: I-IV are meant to indicate
four
stem-loop structures (Been et al., US Patent No. 5,625,047). Hammerhead
Ribozyme: I-
5 III are meant to indicate three stem-loop structures; stems I-III can be of
any length and
may be symmetrical or asymmetrical (Usman et al., 1996, Curr. Op. Struct.
Bio., 1, 527).
Hairpin °bozyme: Helix 1, 4 and 5 can be of any length; Helix 2 is
between 3 and 8
base-pairs long; Y is a pyrimidine; Helix 2 (H2) is provided with a least 4
base pairs (i.e.,
n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more
bases
10 (preferably 3 - 20 bases, i. e., m is from 1 - 20 or more). Helix 2 and
helix 5 may be
covalently linked by one or more bases (i.e., r is >_ 1 base). Helix 1, 4 or S
may also be
extended by 2 or more base pairs (e.g., 4 - 20 base pairs) to stabilize the
ribozyme
structure, and preferably is a protein binding site. In each instance, each N
and N'
independently is any normal or modified base and each dash represents a
potential base-
15 pairing interaction. These nucleotides may be modified at the sugar, base
or phosphate.
Complete base-pairing is not required in the helices, but is preferred. Helix
1 and 4 can be
of any size (i.e., o and p is each independently from 0 to any number, e.g.,
20) as long as
some base-pairing is maintained. Essential bases are shown as specific bases
in the
structure, but those in the art will recognize that one or more may be
modified chemically
(abasic, base, sugar and/or phosphate modifications) or replaced with another
base without
significant effect. Helix 4 can be formed from two separate molecules, i.e.,
without a
connecting loop. The connecting loop when present may be a ribonucleotide with
or
without modifications to its base, sugar or phosphate. "q" >_ is 2 bases. The
connecting
loop can also be replaced with a non-nucleotide linker molecule. H refers to
bases A, U,
or C. Y refers to pyrimidine bases. " " refers to a covalent bond. (Burke et
al., 1996,
Nucleic Acids & Mol. Biol., 10, 129; Chowrira et al., US Patent No.
5,631,359).
Figure 2 shows examples of chemically stabilized ribozyme motifs. HH Rz,
represents hammerhead ribozyme motif (LJsman et al., 1996, Curr. Op. Struct.
Bio., 1,
527); NCH Rz represents the NCH ribozyme motif (described herein and in Ludwig
&
Sproat, International PCT Publication No. WO 98/58058); G-Cleaver, represents
G-
cleaver ribozyme motif (Kore et al., 1998, Nucleic Acids Research, 26, 4116-
4120). N or
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16
n, represent independently a nucleotide which may be same or different and
have
complementarity to each other; rI, represents ribo-Inosine nucleotide; arrow
indicates the
site of cleavage within the target. Position 4 of the HH Rz and the NCH Rz is
shown as
having 2'-C-allyl modification, but those skilled in the art will recognize
that this position
can be modified with other modifications well known in the art, so long as
such
modifications do not significantly inhibit the activity of the ribozyme.
Figure 3 shows an example of the Amberzyme ribozyme motif that is chemically
stabilized (see, for example, Beigelman et al., International PCT publication
No. WO
99/55857; also referred to as Class I Motif). The Amberzyme motif is a class
of enzymatic
nucleic acid molecules that do not require the presence of a ribonucleotide
(2'-OH) group
for activity.
Figure 4 shows an example of the Zinzyme A ribozyme motif that is chemically
stabilized (see, for example, International PCT publication No. WO 99/55857;
also
referred to as Class A Motif). The Zinzyme motif is a class of enzymatic
nucleic acid
molecules that do not require the presence of a ribonucleotide (2'-OH) group
for activity.
Figure 5 shows an example of a DNAzyme motif described by Santoro et al.,
1997,
PNAS, 94, 4262.
Figure 6 is a diagrammatic representation of the hammerhead ribozyme motif
known in the art and the NCH motif. Stem II can be 2 base-pair long,
preferably, 2, 3,
4, 5, 6, 7, 8, and 10 base-pairs long. Each N and N' is independently any base
or non-
nucleotide as used herein; X is adenosine, cytidine or uridine; Stem I-III are
meant to
indicate three stem-loop structures; stems I-III can be of any length and may
be
symmetrical or asymmetrical (Usman et al., 1996, Curr. Op. Struct. Bio., 1,
527); arrow
indicates the site of cleavage in the target RNA; Rz refers to ribozyme; Loop
II may be
present or absent. If Loop II is present it is greater than or equal to three
nucleotides,
preferably four nucleotides. The Loop II sequence is preferably 5'-GAAA-3' or
5'-
GUUA-3' .
Figure 7 shows examples of chemically stabilized ribozyme motifs. HH Rz,
represents hammerhead ribozyme motif (LJsman et al., 1996, Curr. Op. Struct.
Bio., 1,
527); NCH-Inosine ltz represents the NCH ribozyme motif with riboinosine at
15.1
position; NCH-Xylo Rz represents the NCH ribozyme with xylo inosine at 15.1
position.
N or n, represent independently a nucleotide which may be same or different
and may have
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17
complementarity to each other; rI, represents ribo-Inosine nucleotide; xI
represent xylo-
inosine; arrow indicates the site of cleavage within the target. Position 4 of
the HH Rz and
the NCH Rzs is shown as having 2'-C-allyl modification, but those skilled in
the art will
recognize that this position can be modified with other modifications well
known in the
art, so long as such modifications do not significantly inhibit the activity
of the ribozyme.
Figure 8 is a graphical representation of data showing inhibition of cell
proliferation
mediated by NCH and HH ribozymes targeted against HER2/neu/ErbB2 gene.
Untreated,
refers to cells not treated with ribozymes; HH RZ refers to hammerhead
ribozyme; NCX
RZ refers to the NCH ribozymes of the invention; IA refers to catalytically
inactive or
attenuated ribozyme used as a control.
Figure 9 is a schematic diagram of the process for the synthesis of beta-D-
xylofuranosyl hypoxantine 3'-phosphoramidite.
Figure 10 displays a schematic representation of NTP synthesis using
nucleoside
substrates.
Figure 11 shows a scheme for an in vitro selection method. A pool of nucleic
acid
molecules is generated with a random core region and one or more regions) with
a defined
sequence. These nucleic acid molecules are bound to a column containing
immobilized
oligonucleotide with a defined sequence, where the defined sequence is
complementary to
regions) of defined sequence of nucleic acid molecules in the pool. Those
nucleic acid
molecules capable of cleaving the immobilized oligonucleotide (target) in the
column are
isolated and converted to complementary DNA (cDNA), followed by transcription
using
NTPs to form a new nucleic acid pool.
Figure 12 shows a scheme for a two column in vitro selection method. A pool of
nucleic acid molecules is generated with a random core and two flanking
regions (region A
and region B) with defined sequences. The pool is passed through a column
which has
immobilized oligonucleotides with regions A' and B' that are complementary to
regions A
and B of the nucleic acid molecules in the pool, respectively. The column is
subjected to
conditions sufficient to facilitate cleavage of the immobilized
oligonucleotide target. The
molecules in the pool that cleave the target (active molecules) have A' region
of the target
bound to their A region, whereas the B region is free. The column is washed to
isolate the
active molecules with the bound A' region of the target. This pool of active
molecules
may also contain some molecules that are not active to cleave the target
(inactive
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18
molecules) but have dissociated from the column. To separate the contaminating
inactive
molecules from the active molecules, the pool is passed through a second
column (column
2) which contains immobilized oligonucleotides with the A' sequence but not
the B'
sequence. The inactive molecules will bind to column 2 but the active
molecules will not
bind to column 2 because their A region is occupied by the A' region of the
target
oligonucleotide from column 1. Column 2 is washed to isolate the active
molecules for
further processing as described in the scheme shown in Figure 11.
Figure 13 is a diagram of a novel 48 nucleotide enzymatic nucleic acid motif
which
was identified using in vitro methods described in the instant invention. The
molecule
shown is only exemplary. The 5' and 3' terminal nucleotides (referring to the
nucleotides
of the substrate binding arms rather than merely the single terminal
nucleotide on the 5'
and 3' ends) can be varied so long as those portions can base-pair with target
substrate
sequence. In addition, the guanosine (G) shown at the cleavage site of the
substrate can be
changed to other nucleotides so long as the change does not eliminate the
ability of
enzymatic nucleic acid molecules to cleave the target sequence. Substitutions
in the
nucleic acid molecule and/or in the substrate sequence can be readily tested,
for example,
as described herein.
Figure 14 is a schematic diagram of HCV luciferase assay used to demonstrate
efficacy of class I enzymatic nucleic acid molecule motif.
Figure 15 is a graph indicating the dose curve of an enzymatic nucleic acid
molecule
targeting site 146 on HCV RNA.
Figure 16 is a bar graph showing enzymatic nucleic acid molecules targeting 4
sites
within the HCV RNA are able to reduce RNA levels in cells.
Figure 17 shows secondary structures and cleavage rates for characterized
Class II
enzymatic nucleic acid motifs.
Figure 18 is a diagram of a novel 35 nucleotide enzymatic nucleic acid motif
which
was identified using in vitro methods described in the instant invention. The
molecule
shown is only exemplary. The 5' and 3' terminal nucleotides (refernng to the
nucleotides
of the substrate binding arms rather than merely the single terminal
nucleotide on the 5'
and 3' ends) can be varied so long as those portions can base-pair with target
substrate
sequence. In addition, the guanosine (G) shown at the cleavage site of the
substrate can be
changed to other nucleotides so long as the change does not eliminate the
ability of
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19
enzymatic nucleic acid molecules to cleave the target sequence. Substitutions
in the
nucleic acid molecule and/or in the substrate sequence can be readily tested,
for example,
as described herein.
Figure 19 is a bar graph showing substrate specificities for Class II
(zinzyme)
ribozymes.
Figure 20 is a bar graph showing Class II enzymatic nucleic acid molecules
targeting 10 representative sites within the HER2 RNA in a cellular
proliferation screen.
Figure 21 is a synthetic scheme outlining the synthesis of 5-[3-
aminopropynyl(propyl)]uridine 5'-triphosphates and 4-imidazoleaceticacid
conjugates.
Figure 22 is a synthetic scheme outlining the synthesis of 5-[3-(N-4-
imidazoleacetyl)aminopropynyl(propyl)]uridine S'-triphosphates.
Figure 23 is a synthetic scheme outlining the synthesis of carboxylate
tethered
uridine S'-triphosphoates.
Figure 24 is a synthetic scheme outlining the synthesis of S-(3-aminoalkyl)
and 5-
[3(N-succinyl)aminopropyl] functionalized cytidines.
Figure 25 is a diagram of a class I ribozyme stem truncation and loop
replacement
analysis.
Figure 26 is a diagram of class I ribozymes with truncated stems) and/or non-
nucleotide linkers used in loop structures.
Figure 27 is a diagram of "no-ribo" class II ribozymes.
Figure 28 is a graph showing cleavage reactions with class II ribozymes under
differing divalent metal concentrations.
Figure 29 is a diagram of differing class II ribozymes with varying ribo
content and
their relative rates of catalysis.
Figure 30 is a graph showing class II ribozyme (zinzyme) mediated reduction of
HERZ RNA in SKBR3 breast carcinoma cells. Cells were treated with 100 nm, and
200
run of zinzyme (RPI 18656) targeting site 972 of HER2 RNA and a corresponding
scrambled attenuated control complexed with 2.5 p,g/ml of lipid. Active
zinzymes and
scrambled attenuated controls were compared to untreated cells after 24 hours
post
treatment.
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Figure 31 is a graph showing class II ribozyme (zinzyme) mediated dose
response
anti-prolferation assay in SKBR3 breast carcinoma cells. Cells were treated
with 100 nm,
and 200 nm of zinzyme (RPI 18656) targeting site 972 of HER2 RNA and a
corresponding
scrambled attenuated control complexed with 2.0 ~g/ml of lipid. Active
zinzymes and
5 scrambled attenuated controls were compared to untreated cells after 24
hours post
treatment.
Figure 32 is a graph which shows the dose dependent reduction of HER2 RNA in
SKOV-3 cells treated with RPI 19293 from 0 to 100 nM with 5.0 ~g/ml of
cationic lipid.
Figure 33 is a graph which shows the dose dependent reduction of HER2 RNA and
10 inhibition of cellular proliferation in SKBR-3 cells treated with RPI 19293
from 0 to 400
nM with S.0 ~g/ml of cationic lipid.
Figure 34 shows a non-limiting example of the replacement of a 2'-O-methyl 5'-
CA-3' with a ribo G in the class II (zinzyme) motif. The representative motif
shown for
the purpose of the figure is a "seven-ribo" zinzyme motif, however, the
interchangeability
15 of a G and a CA in the position shown in Figure 25 of the class II
(zinzyme) motif extends
to any combination of 2-O-methyl and ribo residues. For instance, a 2'-O-
methyl G can
replace the 2'-O-methyl S'-CA-3' and vise versa.
Figure 35 is a graph which shows a screen of class II ribozymes (zinzymes)
targeting site 972 of HER2 RNA which contain ribo-G reductions (RPI 19727 = no
ribo,
20 RPI 19728 = one ribo, RPI 19293 = two ribo, RPI 19729 = three ribo, RPI
19730 = four
ribo, 19731 = five ribo, and RPI 19292 = seven ribo) for anti-proliferative
activity in
SKBR3 cells.
Figure 36 summarizes the results of functional group modification studies in
which
various nucleoside analogs were tested for activity in the NCH ribozyme motif.
Krel
values describe the cleavage values of a given substituent at position 15.1
relative the
Inosine at position 15.1 (I-15.1).
Figure 37 summarizes reported functional group modification studies performed
at
the A 15.1 residue in the A-15.1 ~U-16.1 context of NUH cleaving ribozymes.
Krel values
describe the cleavage values of a given substituent at position 15.1 relative
the adenosine
at position 15.1 (A-15.1).
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21
Mechanism of action of Nucleic Acid Molecules of the Invention
Antisense: Antisense molecules may be modified or unmodified RNA, DNA, or
mixed polymer oligonucleotides and primarily function by specifically binding
to
matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, Nov
1994,
BioPharm, 20-33). The antisense oligonucleotide binds to target RNA by Watson
Crick
base-pairing and blocks gene expression by preventing ribosomal translation of
the bound
sequences either by steric blocking or by activating RNase H enzyme. Antisense
molecules may also alter protein synthesis by interfering with RNA processing
or transport
from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in
Oncogenesis 7, 151-190).
In addition, binding of single stranded DNA to RNA may result in nuclease
degradation of the heteroduplex (Wu-Pong, supra; Crooke, supra). To date, the
only
backbone modified DNA chemistry which will act as substrates for RNase H are
phosphorothioates, phosphorodithioates, and borontrifluoridates. Recently, it
has been
reported that 2'-arabino and 2'-fluoro arabino- containing oligos can also
activate RNase
H activity.
A number of antisense molecules have been described that utilize novel
configurations of chemically modified nucleotides, secondary structure, and/or
RNase H
substrate domains (Woolf et al., International PCT Publication No. WO
98/13526;
Thompson et al., International PCT Publication No. WO 99/54459 ; Hartmann et
al.,
International PCT Publication No. WO 00/17346) all of these are incorporated
by
reference herein in their entirety.
Antisense DNA can be used to target RNA by means of DNA-RNA interactions,
thereby activating RNase H, which digests the target RNA in the duplex.
Antisense DNA
can be chemically synthesized or can be expressed via the use of a single
stranded DNA
intracellular expression vector or the equivalent thereof.
Triplex Forming OIiQonucleotides (TFO): Single stranded DNA may be designed to
bind to genomic DNA in a sequence specific manner. TFOs are comprised of
pyrimidine-
rich oligonucleotides which bind DNA helices through Hoogsteen Base-pairing
(Wu-Pong,
supra). The resulting triple helix composed of the DNA sense, DNA antisense,
and TFO
disrupts RNA synthesis by RNA polymerase. The TFO mechanism may result in gene
expression or cell death since binding may be irreversible (Mukhopadhyay &
Roth, supra)
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22
2'-5' Oli-og adenylates: The 2-5 A system is an interferon-mediated mechanism
for
RNA degradation found in higher vertebrates (Mitra et al., 1996, Proc Nat Acad
Sci USA
93, 6780-6785). - Two types of enzymes, 2-5A synthetase and RNase L, are
required for
RNA cleavage. The 2-5A synthetases require double stranded RNA to form 2'-5'
oligoadenylates (2-5A). 2-5A then acts as an allosteric effector for utilizing
RNase L
which has the ability to cleave single stranded RNA. The ability to form 2-5A
structures
with double stranded RNA makes this system particularly useful for inhibition
of viral
replication.
(2'-5') oligoadenylate structures may be covalently linked to antisense
molecules to
form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra).
These
molecules putatively bind and activate a 2-5A dependent RNase, the
oligonucleotide/enzyme complex then binds to a target RNA molecule which can
then be
cleaved by the RNase enzyme. The covalent attachment of 2'-5' oligoadenylate
structures
is not limited to antisense applications, and can be further elaborated to
include attachment
to nucleic acid molecules of the instant invention.
Enz~natic Nucleic Acid: Seven basic varieties of naturally-occurnng enzymatic
RNAs are presently known. In addition, several in vitro selection (evolution)
strategies
(Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolve new
nucleic
acid catalysts capable of catalyzing cleavage and ligation of phosphodiester
linkages
(Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641;
Joyce, 1992,
Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel
et a1.,1993,
Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995,
FASEB J., 9,
1183; Breaker, 1996, Curr. Op. Biotech., 7, 442; Santoro et al., 1997, Proc.
Natl. Acad.
Sci., 94, 4262; Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994,
supra; Long &
IJhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish et al., 1997,
Biochemistry 36,
6495; all of these are incorporated by reference herein). Each can catalyze a
series of
reactions including the hydrolysis of phosphodiester bonds in trans (and thus
can cleave
other RNA molecules) under physiological conditions.
In general, enzymatic nucleic acids act by first binding to a target RNA. Such
binding occurs through the target binding portion of an enzymatic nucleic acid
which is
held in close proximity to an enzymatic portion of the molecule that acts to
cleave the
target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a
target
CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
23
RNA through complementary base-pairing, and once bound to the correct site,
acts
enzymatically to cut the target RNA. Strategic cleavage of such a target RNA
will destroy
its ability to direct synthesis of an encoded protein. After an enzymatic
nucleic acid has
bound and cleaved its RNA target, it is released from that RNA to search for
another target
and can repeatedly bind and cleave new targets.
Nucleic acid molecules of this invention will block to some extent PTP-1B,
MetAP-
2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV protein expression and can be
used
to treat disease or diagnose disease associated with the levels of PTP-1B,
MetAP-2,
BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV.
The enzymatic nature of a ribozyme has significant advantages, such as the
concentration of ribozyme necessary to affect a therapeutic treatment is low.
This
advantage reflects the ability of the ribozyme to act enzymatically. Thus, a
single
ribozyme molecule is able to cleave many molecules of target RNA. In addition,
the
ribozyme is a highly specific inhibitor, with the specificity of inhibition
depending not
only on the base-pairing mechanism of binding to the target RNA, but also on
the
mechanism of target RNA cleavage. Single mismatches, or base-substitutions,
near the
site of cleavage can be chosen to completely eliminate catalytic activity of a
ribozyme.
Nucleic acid molecules having an endonuclease enzymatic activity are able to
repeatedly cleave other separate RNA molecules in a nucleotide base sequence-
specific
manner. Such enzymatic nucleic acid molecules can be targeted to virtually any
RNA
transcript, and achieve efficient cleavage in vitro (Zaug et al., 324, Nature,
429 1986 ;
Uhlenbeck, 1987 Nature, 328, 596; Kim et al., 84 Proc. Natl. Acad. Sci. USA,
8788, 1987;
Dreyfus, 1988, Einstein Quart. J. Bio. Med., 6, 92; Haseloff and Gerlach, 334
Nature, 585,
1988; Cech, 260 JAMA, 3030, 1988; Jefferies et al., 17 Nucleic Acids Research,
1371,
1989; and Santoro et al., 1997 supra).
Because of their sequence specificity, trans-cleaving ribozymes show promise
as
therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med.
Chem.
30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037).
Ribozymes
can be designed to cleave specific RNA targets within the background of
cellular RNA.
Such a cleavage event renders the RNA non-functional and abrogates protein
expression
from that RNA. In this manner, synthesis of a protein associated with a
disease state can
be selectively inhibited (Warashina et al., 1999, Chemistry and Biology, 6,
237-250.
CA 02403243 2002-02-21
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24
The nucleic acid molecules of the instant invention are also referred to as
GeneBlocTM reagents, which are essentially nucleic acid molecules (e.g.;
ribozymes,
antisense) capable of down-regulating gene expression.
Tar-eg t sites
Targets for useful ribozymes and antisense nucleic acids can be determined as
disclosed in Draper et al., WO 93/23569; Sullivan et al., WO 93/23057;
Thompson et al.,
WO 94/02595; Draper et al., WO 95/04818; McSwiggen et al., US Patent No.
5,525,468,
and all hereby incorporated in their entireties by reference herein. Other
examples include
the following PCT applications, which concern inactivation of expression of
disease-
related genes: WO 95/23225, WO 95/13380, WO 94/02595, all incorporated by
reference
herein. Rather than repeat the guidance provided in those documents here,
below are
provided specific examples of such methods, not limiting to those in the art.
Ribozymes
and antisense to such targets are designed as described in those applications
and
synthesized to be tested in vitro and in vivo, as also described. The sequence
of human
PTP-1B, MetAP-2, BACE, ps-1, ps-2, HER2, PLN, TERT, and/or HBV RNAs (for
example, GenBank accession Nos. (PTP-1B,. NM_002827), (MetAP-2, U29607),
(BACE,
AF190725), (ps-1, L76517), (ps-2, L43964), (HER2/c-erb2/neu, X03363), (PLN,
NM_002667), (TERT, NM_003219) and (HBV, AF100308.1, HBV strain 2-18;
additionally, other HBV strains can be screened by one skilled in the art, see
Table 35 for
other possible strains) were screened for optimal enzymatic nucleic acid and
antisense
target sites using a computer-folding algorithm. Antisense, hammerhead,
DNAzyme,
NCH (Inozyme), amberzyme, zinzyme or G-Cleaver ribozyme binding/cleavage sites
were
identified. These sites are shown in Tables 3-29, 31, 33, 34, 37-43, 56, 58,
59, 62, 63 (all
sequences are S' to 3' in the tables; X can be any base-paired sequence, the
actual
sequence is not relevant here). The nucleotide base position is noted in the
Tables as that
site to be cleaved by the designated type of enzymatic nucleic acid molecule.
Table 36
shows substrate positions selected from Renbo et al., 1987, Sci. Sin., 30,
507, used in
Draper, US patent No. 6,017,756 entitled "METHOD AND REAGENT FOR
INHIBITING HEPATITIS B VIRUS REPLICATION" and Draper et al., International
PCT publication No. WO 93/23569, filed April 29, 1993, entitled "METHOD AND
REAGENT FOR INHIBITING VIRAL REPLICATION". While human sequences can be
CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
screened and enzymatic nucleic acid molecule and/or antisense thereafter
designed, as
discussed in Stinchcomb et al., WO 95/23225, mouse targeted ribozymes may be
useful to
test efficacy of action of the enzymatic nucleic acid molecule and/or
antisense prior to
testing in humans.
5 Antisense, hammerhead, DNAzyme, NCH (Inozyme), amberzyme, zinzyme or G-
Cleaver ribozyme binding/cleavage sites were identified, as discussed above.
The nucleic
acid molecules were individually analyzed by computer folding (Jaeger et al.,
1989 Proc.
Natl. Acad. Sci. USA, 86, 7706) to assess whether the sequences fold into the
appropriate
secondary structure. Those nucleic acid molecules with unfavorable
intramolecular
10 interactions such as between the binding arms and the catalytic core were
eliminated from
consideration. Varying binding arm lengths can be chosen to optimize activity.
Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver
ribozyme binding/cleavage sites were identified and were designed to anneal to
various
sites in the RNA target. The binding arms are complementary to the target site
sequences
15 described above. The nucleic acid molecules were chemically synthesized.
The method of
synthesis used follows the procedure for normal DNA/RNA synthesis as described
below
and in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990
Nucleic
Acids Res., 18, 5433; Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684;
and
Caruthers et al., 1992, Methods in Enzymology 211,3-19.
Synthesis of Nucleic acid Molecules
Synthesis of nucleic acids greater than 100 nucleotides in length is difficult
using
automated methods, and the therapeutic cost of such molecules is prohibitive.
In this
invention, small nucleic acid motifs ("small refers to nucleic acid motifs no
more than 100
nucleotides in length, preferably no more than 80 nucleotides in length, and
most
preferably no more than 50 nucleotides in length; e.g., antisense
oligonucleotides,
hammerhead or the NCH ribozymes) are preferably used for exogenous delivery.
The
simple structure of these molecules increases the ability of the nucleic acid
to invade
targeted regions of RNA structure. Exemplary molecules of the instant
invention are
chemically synthesized, and others can similarly be synthesized.
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26
Oligonucleotides (e.g.; antisense GeneBlocs) are synthesized using protocols
known
in the art as described in Caruthers et al., 1992, Methods in Enzymology 211,
3-19,
Thompson et al., International PCT Publication No. WO 99/54459, Wincott et
al., 1995,
Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74,
59,
Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, US patent
No.
6,001,311. All of these references are incorporated herein by reference. The
synthesis of
oligonucleotides makes use of common nucleic acid protecting and coupling
groups, such
as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a non-
limiting
example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc.
synthesizer using a 0.2 ~mol scale protocol with a 2.5 min coupling step for
2'-O-
methylated nucleotides and a 45 sec coupling step for 2'-deoxy nucleotides.
Table II
outlines the amounts and the contact times of the reagents used in the
synthesis cycle.
Alternatively, syntheses at the 0.2 pmol scale can be performed on a 96-well
plate
synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with
minimal
modification to the cycle. A 33-fold excess (60 pL of 0.11 M = 6.6 ~,mol) of
2'-O-methyl
phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 pL of 0.25 M =
15 pmol)
can be used in each coupling cycle of 2'-O-methyl residues relative to polymer-
bound S'-
hydroxyl. A 22-fold excess (40 p.L of 0.11 M = 4.4 ~mol) of deoxy
phosphoramidite and a
70-fold excess of S-ethyl tetrazole (40 pL of 0.25 M =10 pmol) can be used in
each
coupling cycle of deoxy residues relative to polymer-bound 5'-hydroxyl.
Average
coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by
colorimetric quantitation of the trityl fractions, are typically 97.5-99%.
Other
oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc.
synthesizer
include the following: detritylation solution is 3% TCA in methylene chloride
(ABI);
capping is performed with 16% N methyl imidazole in THF (ABI) and 10% acetic
anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I2,
49 mM
pyridine, 9% water in THF (PERSEPTIVETM). Burdick & Jackson Synthesis Grade
acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole
solution (0.25 M in
acetonitrile) is made up from the solid obtained from American International
Chemical,
Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage
reagent (3H-
1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.
CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
27
Deprotection of the antisense oligonucleotides is performed as follows: the
polymer-
bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top
vial and
suspended in a solution of 40% aq. methylamine (1 mL) at 65 °C for 10
min. After
cooling to -20 °C, the supernatant is removed from the polymer support.
The support is
washed three times with 1.0 mL of EtOH:MeCN:H20/3:1:1, vortexed and the
supernatant
is then added to the first supernatant. The combined supernatants, containing
the
oligoribonucleotide, are dried to a white powder.
The method of synthesis used for normal RNA including certain enzymatic
nucleic
acid molecules follows the procedure as described in Usman et al., 1987, J.
Am. Chem.
Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and
Wincott et al.,
1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol.
Bio., 74, 59,
and makes use of common nucleic acid protecting and coupling groups, such as
dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a non-
limiting
example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc.
synthesizer using a 0.2 pmol scale protocol with a 7.5 min coupling step for
alkylsilyl
protected nucleotides and a 2.5 min coupling step for 2'-O-methylated
nucleotides. Table
II outlines the amounts and the contact times of the reagents used in the
synthesis cycle.
Alternatively, syntheses at the 0.2 ~mol scale can be done on a 96-well plate
synthesizer,
such as the instrument produced by Protogene (Palo Alto, CA) with minimal
modification
to the cycle. A 33-fold excess (60 ~L of 0.11 M = 6.6 ~mol) of 2'-O-methyl
phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 ~L of 0.25 M =15
pmol)
can be used in each coupling cycle of 2'-O-methyl residues relative to polymer-
bound S'-
hydroxyl. A 66-fold excess (120 ~L of 0.11 M = 13.2 ~mol) of alkylsilyl (ribo)
protected
phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 ~L of 0.25 M =
30 pmol)
can be used in each coupling cycle of ribo residues relative to polymer-bound
5'-hydroxyl.
Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,
determined by
colorimetric quantitation of the trityl fractions, are typically 97.5-99%.
Other
oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc.
synthesizer
include the following: detritylation solution is 3% TCA in methylene chloride
(ABI);
capping is performed with 16% N methyl imidazole in THF (ABn and 10% acetic
anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I2, 49
mM
pyridine, 9% water in THF (PERSEPTIVETM). Burdick & Jackson Synthesis Grade
CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
28
acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole
solution (0.25 M in
acetonitrile) is made up from the solid obtained from American International
Chemical,
Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage
reagent (3H-
1,2-Benzodithiol-3-one 1,1-dioxide0.05 M in acetonitrile) is used.
Deprotection of the RNA is performed using either a two-pot or one-pot
protocol.
For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is
transferred to
a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine
(1 mL) at
65 °C for 10 min. After cooling to -20 °C, the supernatant is
removed from the polymer
support. The support is washed three times with 1.0 mL of EtOH:MeCN:H20/3:1:1,
vortexed and the supernatant is then added to the first supernatant. The
combined
supernatants, containing the oligoribonucleotide, are dried to a white powder.
The base
deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP
solution (300
pL of a solution of 1.5 mL N-methylpyrrolidinone, 750 ~L TEA and 1 mL TEA~3HF
to
provide a 1.4 M HF concentration) and heated to 65 °C. After 1.5 h, the
oligomer is
quenched with 1.5 M NH4HC03.
Alternatively, for the one-pot protocol, the polymer-bound trityl-on
oligoribonucleotide is transferred to a 4 mL glass screw top vial and
suspended in a
solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65 °C for
15 min. The
vial is brought to r.t. TEA~3HF (0.1 mL) is added and the vial is heated at 65
°C for 1 S
min. The sample is cooled at -20 °C and then quenched with 1.5 M
NH4HC03.
For purification of the trityl-on oligomers, the quenched NH4HC03 solution is
loaded onto a C-18 containing cartridge that had been prewashed with
acetonitrile
followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA
is
detritylated with 0.5% TFA for 13 min. The cartridge is then washed again with
water,
salt exchanged with 1 M NaCI and washed with water again. The oligonucleotide
is then
eluted with 30% acetonitrile.
Inactive hammerhead ribozymes or binding attenuated control (BAC)
oligonucleotides) are synthesized by substituting a U for GS and a U for A14
(numbering
from Hertel, K. J., et al., 1992, Nucleic Acids Res., 20, 3252). Similarly,
one or more
nucleotide substitutions can be introduced in other enzymatic nucleic acid
molecules to
inactivate the molecule and such molecules can serve as a negative control.
CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
29
The average stepwise coupling yields are typically >98% (Wincott et al., 1995
Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will
recognize that
the scale of synthesis can be adapted to be larger or smaller than the example
described
above including but not limited to 96-well format, all that is important is
the ratio of
chemicals used in the reaction.
Alternatively, the nucleic acid molecules of the present invention can be
synthesized
separately and joined together post-synthetically, for example, by ligation
(Moore et al.,
1992, Science 256, 9923; Draper et al., International PCT publication No. WO
93/23569;
Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997,
Nucleosides
& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204).
The nucleic acid molecules of the present invention are modified extensively
to
enhance stability by modification with nuclease resistant groups, for example,
2'-amino, 2'-
C-allyl, 2'-flouro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren,
1992, TIBS
17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). Ribozymes are
purified by
gel electrophoresis using general methods or are purified by high pressure
liquid
chromatography (HPLC; see Wincott et al., supra, the totality of which is
hereby
incorporated herein by reference) and are re-suspended in water.
The sequences of the ribozymes and antisense constructs that are chemically
synthesized, useful in this study, are shown in Tables 3-31, 33, 34, 37-43,
56, 58, 59, 62,
63. Those in the art will recognize that these sequences are representative
only of many
more such sequences where the enzymatic portion of the ribozyme (all but the
binding
arms) is altered to affect activity. The ribozyme and antisense construct
sequences listed in
Tables 3-31, 33, 34, 37-43, 56, 58, 59, 62, 63 may be formed of
ribonucleotides or other
nucleotides or non-nucleotides. Such ribozymes with enzymatic activity are
equivalent to
the ribozymes described specifically in the Tables.
timizing_Activity of the nucleic acid molecule of the invention.
Chemically synthesizing nucleic acid molecules with modifications (base, sugar
and/or phosphate) that prevent their degradation by serum ribonucleases may
increase their
potency (see e.g., Eckstein et al., International Publication No. WO 92/07065;
Perrault et
al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and
Cedergren,
1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication
No. WO
93/15187; Rossi et al., International Publication No. WO 91/03162; Sproat, US
Patent No.
CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
5,334,711; and Burgin et al., supra; all of these describe various chemical
modifications
that can be made to the base, phosphate and/or sugar moieties of the nucleic
acid
molecules herein and are all hereby incorporated by reference herein).
Modifications
which enhance their efficacy in cells, and removal of bases from nucleic acid
molecules to
5 shorten oligonucleotide synthesis times and reduce chemical requirements are
desired.
There are several examples in the art describing sugar, base and phosphate
modifications that can be introduced into nucleic acid molecules (e.g.,
enzymatic nucleic
acid molecules) without significantly effecting catalysis and with significant
enhancement
in their nuclease stability and efficacy. Enzymatic nucleic acid molecules are
modified to
10 enhance stability and/or enhance catalytic activity by modification with
nuclease resistant
groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-D-methyl, 2'-O-allyl,
2'-H,
nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS
17, 34;
Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996
Biochemistry 35,
14090). Sugar modification of enzymatic nucleic acid molecules have been
extensively
15 described in the art (see Eckstein et al., International Publication PCT
No. WO 92/07065;
Perrault et al. Nature 1990, 344, 565-568; Pieken et al. Science 1991, 253,
314-317;
Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman et al.
International Publication PCT No. WO 93/15187; Sproat, US Patent No. 5,334,711
and
Beigelman et al., 1995 J. Biol. Chem. 270, 25702; all of the references are
hereby
20 incorporated in their totality by reference herein). Such publications
describe general
methods and strategies to determine the location of incorporation of sugar,
base and/or
phosphate modifications and the like into enzymatic nucleic acid molecules
without
inhibiting catalysis, and are incorporated by reference herein. The 2'-
position of the sugar
in a nucleotide present in the nucleic acid molecules of the instant invention
which
25 tolerates substitution is selected from the group comprising -H, -OH, -
COOH, -CONHz, -
CONHR~, -CONR1R2, -NH2, -NHR~, -NR~R2, -NHCOR~, -SH, SRI, -F, -ONH2, -
ONHRI, -ONR1R2, -NHOH, -NHORI, -NRzOH, -NRZORI, substituted or unsubstituted
C~-Clo straight chain or branched alkyl, substituted or unsubstituted C2-Coo
straight chain
or branched alkenyl, substituted or unsubstituted CZ-Clo straight chain or
branched alkynyl,
30 substituted or unsubstituted C1-Coo straight chain or branched alkoxy,
substituted or
unsubstituted Cz-Clo straight chain or branched alkenyloxy, and substituted or
unsubstituted C2-Clo straight chain or branched alkynyloxy. The substituents
for sugar 2'
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31
position preferably are independently halogen, cyano, amino, carboxy, ester,
ether,
carboxamide, hydroxy, or mercapto. R1 and RZ can be substituted or
unsubstituted alkyl,
alkenyl, or alkynyl groups, where the substituents are independently halogen,
cyano,
amino, carboxy, ester, ether, carboxamide, hydroxy, or mercapto.
In view of such teachings, similar modifications can be used as described
herein to
modify the nucleic acid molecules of the instant invention. Such publications
describe
general methods and strategies to determine the location of incorporation of
sugar, base
and/or phosphate modifications and the like into ribozymes without inhibiting
catalysis,
and are incorporated by reference herein. In view of such teachings, similar
modifications
can be used as described herein to modify the nucleic acid molecules of the
instant
invention.
Some of the non-limiting examples of base modifications that can be introduced
into
enzymatic nucleic acids without significantly effecting their catalytic
activity include,
inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-
trimethoxy
benzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-
alkylcytidines (e.g.,
S-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,
5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-
methyluridine) and
others (Burgin et al., 1996, Biochemistry, 35, 14090). By "modified bases" in
this aspect is
meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1'
position or
their equivalents; such bases may be used within the catalytic core of the
enzyme and/or in
the substrate-binding regions.
The nucleic acid bases can be hypoxanthin-9-yl, or a functional equivalent
thereof,
in positionl5vof the ribozyme; the base at other positions may be guanin-9-yl,
hypoxanthin-9-yl or 7-deazaguanin-9-yl in positions 5, 8 and 12 in the
ribozyme; adenin-9-
y1, 2,6-diaminopurin-9-yl, purin-9-yl or 7-deaza adenin-9-yl in positions 6,
9, 13 and 14;
uracil-1-yl, uracil-S-yl, thymin-1-yl or 5-propynyluracil-1-yl in position 4;
cytosin-1-yl, 5-
methylcytosin-1-yl or 5-propynylcytosin-1-yl in position 3; and adenin-9-yl,
cytosin-1-yl,
guanin-9-yl, uracil-1-yl, uracil-5-yl, hypoxanthin-9-yl, thymin-1-yl, 5-
methylcytosin-1-yl,
2,6-diaminopurin-9-yl, purin-9-yl, 7-deaza adenin-9-yl, 7-deazaguanin-9-yl, 5-
propynylcytosin-1-yl, S-propynyluracil-1-yl, isoguanin-9-yl, 2-aminopurin-9-
yl, 6-
methyluracil-1-yl, 4-thiouracil-1-yl, 2-pyrimidone-1-yl, quinazoline-2,4-dione-
1-yl,
xanthin-9-yl, N2-dimethylguanin-9-yl, or a functional equivalent thereof in
position 7. The
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32
base at position 15.1 is preferably hypoxanthin-9-yl or an analog where no
hydrogen bond
can form between any group at the 2 position of the base and the 2-oxo group
of 016.1.
Preferably, B is not guanin-9-yl in position 15.1.
In particular, the invention features modified ribozymes having a base
substitution
selected from pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-
trimethoxy
benzene, 3-methyluracil, dihydrouracil, naphthyl, 6-methyl-uracil and
aminophenyl.
While chemical modification of oligonucleotide internucleotide linkages with
phosphorothioate, phosphorothioate, and/or S'-methylphosphonate linkages
improves
stability, too many of these modifications may cause some toxicity. Therefore,
when
designing nucleic acid molecules, the amount of these internucleotide linkages
should be
minimized. The reduction in the concentration of these linkages should lower
toxicity
resulting in increased efficacy and higher specificity of these molecules.
Nucleic acid molecules having chemical modifications which maintain or enhance
activity are provided. Such nucleic acid molecules are also generally more
resistant to
nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the
activity may not
be significantly lowered. Therapeutic nucleic acid molecules delivered
exogenously must
optimally be stable within cells until translation of the target RNA has been
inhibited long
enough to reduce the levels of the undesirable protein. This period of time
varies between
hours to days depending upon the disease state. Clearly, nucleic acid
molecules must be
resistant to nucleases in order to function as effective intracellular
therapeutic agents.
Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995
Nucleic
Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211,3-19
(all are
incorporated by reference herein) have expanded the ability to modify nucleic
acid
molecules by introducing nucleotide modifications to enhance their nuclease
stability as
described above.
Use of these the nucleic acid-based molecules of the invention will lead to
better
treatment of the disease progression by affording the possibility of
combination therapies
(e.g., multiple antisense or enzymatic nucleic acid molecules targeted to
different genes,
nucleic acid molecules coupled with known small molecule inhibitors, or
intermittent
treatment with combinations of molecules (including different motifs) and/or
other
chemical or biological molecules). The treatment of patients with nucleic acid
molecules
may also include combinations of different types of nucleic acid molecules.
CA 02403243 2002-02-21
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33
Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acid molecules and
antisense nucleic acid molecules) delivered exogenously must optimally be
stable within
cells until translation of the target RNA has been inhibited long enough to
reduce the
levels of the undesirable protein. This period of time varies between hours to
days
depending upon the disease state. Clearly, these nucleic acid molecules must
be resistant
to nucleases in order to function as effective intracellular therapeutic
agents.
Improvements in the chemical synthesis of nucleic acid molecules described in
the instant
invention and in the art have expanded the ability to modify nucleic acid
molecules by
introducing nucleotide modifications to enhance their nuclease stability as
described
above.
By "enhanced enzymatic activity" is meant to include activity measured in
cells
and/or in vivo where the activity is a reflection of both catalytic activity
and ribozyme
stability. In this invention, the product of these properties is increased or
not significantly
(less than 10-fold) decreased in vivo compared to an all RNA ribozyme or all
DNA
enzyme.
In yet another preferred embodiment, nucleic acid catalysts having chemical
modifications which maintain or enhance enzymatic activity are provided. Such
nucleic
acid catalysts are also generally more resistant to nucleases than unmodified
nucleic acid.
Thus, in a cell and/or in vivo the activity may not be significantly lowered.
As exemplified
herein such ribozymes are useful in a cell and/or in vivo even if activity
over all is reduced
10 fold (Burgin et al., 1996, Biochemistry, 35, 14090). Such ribozymes herein
are said to
"maintain" the enzymatic activity of an all RNA ribozyme.
In another aspect the nucleic acid molecules comprise a S' and/or a 3'- cap
structure.
By "cap structure" is meant chemical modifications, which have been
incorporated
at either terminus of the oligonucleotide (see, for example, Wincott et al.,
WO 97/26270,
incorporated by reference herein). These terminal modifications protect the
nucleic acid
molecule from exonuclease degradation, and may help in delivery and/or
localization
within a cell. The cap may be present at the 5'-terminus (5'-cap) or at the 3'-
terminal (3'-
cap) or may be present on both termini. In non-limiting examples: the 5'-cap
is selected
from the group comprising inverted abasic residue (moiety); 4',5'-methylene
nucleotide; 1-
(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic
nucleotide; 1,5-
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34
anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base
nucleotide;
phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-
seco nucleotide;
acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,
3'-3'-
inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted
nucleotide moiety;
3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate;
hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate;
phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for
more
details, see Wincott et al., International PCT publication No. WO 97/26270,
incorporated
by reference herein).
In yet another preferred embodiment, the 3'-cap is selected from a group
comprising, 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl)
nucleotide; 4'-thin
nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-
propyl
phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl
phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-
nucleotide; alpha-
nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl
nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-
dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'-inverted
abasic moiety;
5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino;
bridging
and/or non-bridging 5'-phosphoramidate, phosphorothioate and/or
phosphorodithioate,
bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more
details
see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference
herein).
An "alkyl" group refers to a saturated aliphatic hydrocarbon, including
straight-
chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group
has 1 to 12
carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more
preferably 1 to 4
carbons. The alkyl group may be substituted or unsubstituted. When substituted
the
substituted groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S, N02 or
N(CH3)2,
amino, or SH. The term also includes alkenyl groups which are unsaturated
hydrocarbon
groups containing at least one carbon-carbon double bond, including straight-
chain,
branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12
carbons.
More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably
1 to 4
carbons. The alkenyl group may be substituted or unsubstituted. When
substituted the
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substituted groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S, N02,
halogen,
N(CH3)2, amino, or SH. The term "alkyl" also includes alkynyl groups which
have an
unsaturated hydrocarbon group containing at least one carbon-carbon triple
bond,
including straight-chain, branched-chain, and cyclic groups. Preferably, the
alkynyl group
5 has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7
carbons, more
preferably 1 to 4 carbons. The alkynyl group may be substituted or
unsubstituted. When
substituted the substituted groups) is preferably, hydroxyl, cyano, alkoxy,
=O, =S, N02
or N(CH3)2, amino or SH.
Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl,
heterocyclic
10 aryl, amide and ester groups. An "aryl" group refers to an aromatic group
which has at
least one ring having a conjugated pi electron system and includes carbocyclic
aryl,
heterocyclic aryl and biaryl groups, all of which may be optionally
substituted. The
preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl,
SH, OH,
cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group
refers to an
15 alkyl group (as described above) covalently joined to an aryl group (as
described above).
Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring
are all
carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl
groups are
groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and
the
remainder of the ring atoms are carbon atoms. Suitable heteroatoms include
oxygen,
20 sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-
lower alkyl pyrrolo,
pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An
"amide" refers
to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen. An
"ester" refers to
an -C(O)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
By "nucleotide" as used herein is as recognized in the art to include .natural
bases
25 (standard), and modified bases well known in the art. Such bases are
generally located at
the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a
base, sugar
and a phosphate group. The nucleotides can be unmodified or modified at the
sugar,
phosphate and/or base moiety, (also referred to interchangeably as nucleotide
analogs,
modified nucleotides, non-natural nucleotides, non-standard nucleotides and
other; see, for
30 example, Usman and McSwiggen, supra; Eckstein et al., International PCT
Publication
No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187;
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36
Uhlman & Peyman, supra, all are hereby incorporated by reference herein).
There are
several examples of modified nucleic acid bases known in the art as summarized
by
Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting
examples of
base modifications that can be introduced into nucleic acid molecules include,
inosine,
purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy
benzene, 3-
methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g.,
S-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,
5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-
methyluridine),
propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman &
Peyman,
supra).
By "modified bases" in this aspect is meant nucleotide bases other than
adenine,
guanine, cytosine and uracil at 1' position or their equivalents; such bases
may be used at
any position, for example, within the catalytic core of an enzymatic nucleic
acid molecule
and/or in the substrate-binding regions of the nucleic acid molecule. Such
modified
nucleotides include dideoxynucleotides which have pharmaceutical utility well
known in
the art, as well as utility in basic molecular biology methods such as
sequencing.
In a preferred embodiment, the invention features modified ribozymes with
phosphate backbone modifications comprising one or more phosphorothioate,
phosphorodithioate, methylphosphonate, morpholino, amidate carbamate,
carboxymethyl,
acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,
thioformacetal,
and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone
modifications,
see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and
Properties, in
Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel
Backbone
Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense
Research,
ACS, 24-39. These references are hereby incorporated by reference herein.
By "abasic" is meant sugar moieties lacking a base or having other chemical
groups
in place of a base at the 1' position, (for more details, see Wincott et al.,
International PCT
publication No. WO 97/26270).
By "unmodified nucleoside" or "unmodified nucleotide" is meant one of the
bases
adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon of (3-D-
ribo-furanose.
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37
By "modified nucleoside" or "modified nucleotide" is meant any nucleotide base
which contains a modification in the chemical structure of an unmodified
nucleotide base,
sugar and/or phosphate.
In connection with 2'-modified nucleotides as described for the present
invention,
by "amino" is meant 2'-NHZ or 2'-O- NH2, which may be modified or unmodif ed.
Such
modified groups are described, for example, in Eckstein et al., U.S. Patent
5,672,695 and
Matulic-Adamic et al., WO 98/28317, which are both incorporated by reference
in their
entireties.
Various modifications to nucleic acid (e.g., antisense and ribozyme) structure
can be
made to enhance the utility of these molecules. Such modifications will
enhance shelf life,
half life in vitro, stability, and ease of introduction of such
oligonucleotides to the target
site, e.g., to enhance penetration of cellular membranes, and confer the
ability to recognize
and bind to targeted cells.
Use of these molecules will lead to better treatment of the disease
progression by
affording the possibility of combination therapies (e.g., multiple ribozymes
targeted to
different genes, ribozymes coupled with known small molecule inhibitors, or
intermittent
treatment with combinations of ribozymes (including different ribozyme motifs)
and/or
other chemical or biological molecules). The treatment of patients with
nucleic acid
molecules may also include combinations of different types of nucleic acid
molecules.
Therapies may be devised which include a mixture of ribozymes (including
different
ribozyme motifs), antisense and/or 2-SA chimera molecules to one or more
targets to
alleviate symptoms of a disease.
Administration of Nucleic Acid Molecules
Methods for the delivery of nucleic acid molecules are described in Akhtar et
al.,
1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense
Oligonucleotide
Therapeutics, ed. Akhtar, 1995 which are both incorporated herein by
reference. Sullivan
et al., PCT WO 94/02595, further describes the general methods for delivery of
enzymatic
RNA molecules. These protocols may be utilized for the delivery of virtually
any nucleic
acid molecule. Nucleic acid molecules may be administered to cells by a
variety of
methods known to those familiar to the art, including, but not restricted to,
encapsulation
in liposomes, by iontophoresis, or by incorporation into other vehicles, such
as hydrogels,
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38
cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For
some
indications, nucleic acid molecules may be directly delivered ex vivo to cells
or tissues
with or without the aforementioned vehicles. Alternatively, the nucleic
acidlvehicle
combination is locally delivered by direct injection or by use of a catheter,
infusion pump
or stmt. Many examples in the art describe CNS delivery methods of
oligonucleotides by
osmotic pump, (see Chun et al., 1998, Neuroscience Letters, 257, 135-138,
D'Aldin et al.,
1998, Mol. Brain Research, 55, 151-164, Dryden et al., 1998, J. Endocrinol.,
157, 169-
175, Ghirnikar et al., 1998, Neuroscience Letters, 247, 21-24) or direct
infusion (Broaddus
et al., 1997, Neurosurg. Focus, 3, article 4). Other routes of delivery
include, but are not
limited to oral (tablet or pill form) and/or intrathecal delivery (Gold, 1997,
Neuroscience,
76, 1153-1158). For a comprehensive review on drug delivery strategies
including broad
coverage of CNS delivery, see Jain, Drug Delivery Systems: Technologies and
Commercial Opportunities, Decision Resources, 1998. Other routes of delivery
include,
but are not limited to, intravascular, intramuscular, subcutaneous or joint
injection, aerosol
inhalation, oral (tablet or pill form), topical, systemic, ocular,
intraperitoneal and/or
intrathecal delivery. More detailed descriptions of nucleic acid delivery and
administration are provided in Sullivan et al., supra, Draper et al., PCT
W093/23569;
Beigelman et al., PCT W099/05094, and Klimuk et al., PCT W099/04819 all of
which
are incorporated by reference herein.
The molecules of the instant invention can be used as pharmaceutical agents.
Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a
symptom to
some extent, preferably all of the symptoms) of a disease state in a patient.
The negatively charged polynucleotides of the invention can be administered
(e.g.,
RNA, DNA or protein) and introduced into a patient by any standard means, with
or
without stabilizers, buffers, and the like, to form a pharmaceutical
composition. When it is
desired to use a liposome delivery mechanism, standard protocols for formation
of
liposomes can be followed. The compositions of the present invention may also
be
formulated and used as tablets, capsules or elixirs for oral administration;
suppositories for
rectal administration; sterile solutions; suspensions for injectable
administration; and the
other compositions known in the art.
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39
The present invention also includes pharmaceutically acceptable formulations
of the
compounds described. These formulations include salts of the above compounds,
e.g.,
acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic
acid, and
benzene sulfonic acid.
A pharmacological composition or formulation refers to a composition or
formulation in a form suitable for administration, e.g., systemic
administration, into a cell
or patient, preferably a human. Suitable forms, in part, depend upon the use
or the route of
entry, for example, oral, transdermal, or by injection. Such forms should not
prevent the
composition or formulation from reaching a target cell (i.e., a cell to which
the negatively
charged polymer is desired to be delivered to). For example, pharmacological
compositions injected into the blood stream should be soluble. Other factors
are known in
the art, and include considerations such as toxicity and forms which prevent
the
composition or formulation from exerting its effect.
By "systemic administration" is meant in vivo systemic absorption or
accumulation
of drugs in the blood stream followed by distribution throughout the entire
body.
Administration routes which lead to systemic absorption include, without
limitations:
intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary
and
intramuscular. Each of these administration routes expose the desired
negatively charged
polymers, e.g., nucleic acids, to an accessible diseased tissue. The rate of
entry of a drug
into the circulation has been shown to be a function of molecular weight or
size. The use
of a liposome or other drug Garner comprising the compounds of the instant
invention can
potentially localize the drug, for example, in certain tissue types, such as
the tissues of the
reticular endothelial system (RES). A liposome formulation which can
facilitate the
association of drug with the surface of cells, such as, lymphocytes and
macrophages is also
useful. This approach may provide enhanced delivery of the drug to target
cells by taking
advantage of the specificity of macrophage and lymphocyte immune recognition
of
abnormal cells, such as cancer cells.
By pharmaceutically acceptable formulation is meant, a composition or
formulation
that allows for the effective distribution of the nucleic acid molecules of
the instant
invention in the physical location most suitable for their desired activity.
Nonlimiting
examples of agents suitable for formulation with the nucleic acid molecules of
the instant
invention include: P-glycoprotein inhibitors (such as Pluronic P85) which can
enhance
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WO 01/16312 PCT/US00/23998
entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin.
Pharmacol.,
13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide)
microspheres
for sustained release delivery after intracerebral implantation (Emerich, DF
et al, 1999,
Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, MA; and loaded
nanoparticles, such
5 as those made of polybutylcyanoacrylate, which can deliver drugs across the
blood brain
burner and can alter neuronal uptake mechanisms (frog Neuropsychopharmacol
Biol .
Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery
strategies for the
nucleic acid molecules of the instant invention include material described in
Boado et al.,
1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-
284;
10 Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug
Delivery Rev.,
15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916;
and Tyler et
al., 1999, PNAS USA., 96, 7053-7058.
The invention also features the use of the composition comprising surface-
modified
liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-
circulating
15 liposomes or stealth liposomes). These formulations offer a method for
increasing the
accumulation of drugs in target tissues. This class of drug carriers resists
opsonization and
elimination by the mononuclear phagocytic system (MPS or RES), thereby
enabling longer
blood circulation times and enhanced tissue exposure for the encapsulated drug
(Lasic et
al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995,
43, 1005-
20 1011 ). Such liposomes have been shown to accumulate selectively in tumors,
presumably
by extravasation and capture in the neovascularized target tissues (Lasic et
al., Science
1995, 267, 1275-1276; Oku et a1.,1995, Biochim. Biophys. Acta, 1238, 86-90).
The long-
circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA
and
RNA, particularly compared to conventional cationic liposomes which are known
to
25 accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42,
24864-24870; Choi
et al., International PCT Publication No. WO 96/10391; Ansell et al.,
International PCT
Publication No. WO 96/10390; Holland et al., International PCT Publication No.
WO
96/10392; all of which are incorporated herein by reference). Long-circulating
liposomes
are also likely to protect drugs from nuclease degradation to a greater extent
compared to
30 cationic liposomes, based on their ability to avoid accumulation in
metabolically
aggressive MPS tissues such as the liver and spleen.
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41
The present invention also includes compositions prepared for storage or
administration which include a pharmaceutically effective amount of the
desired
compounds in a pharmaceutically acceptable carrier or diluent. Acceptable
Garners or
diluents for therapeutic use are well known in the pharmaceutical art, and are
described,
for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R.
Gennaro edit. 1985) hereby incorporated by reference herein. For example,
preservatives,
stabilizers, dyes and flavoring agents may be provided. These include sodium
benzoate,
sorbic acid and esters ofp-hydroxybenzoic acid. In addition, antioxidants and
suspending
agents may be used.
A pharmaceutically effective dose is that dose required to prevent, inhibit
the
occurrence, or treat (alleviate a symptom to some extent, preferably all of
the symptoms)
of a disease state. The pharmaceutically effective dose depends on the type of
disease, the
composition used, the route of administration, the type of mammal being
treated, the
physical characteristics of the specific mammal under consideration,
concurrent
medication, and other factors which those skilled in the medical arts will
recognize.
Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active
ingredients is administered dependent upon potency of the negatively charged
polymer.
The nucleic acid molecules of the present invention may also be administered
to a
patient in combination with other therapeutic compounds to increase the
overall
therapeutic effect. The use of multiple compounds to treat an indication may
increase the
beneficial effects while reducing the presence of side effects.
Alternatively, certain of the nucleic acid molecules of the instant invention
can be
expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub,
1985,
Science, -229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA
83, 399;
Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet
et al., 1992,
Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41;
Weerasinghe et
al., 1991, J. Yirol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci.
USA, 89,
10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al.,
1990 Science,
247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et
al., 1997,
Gene Therapy, 4, 45; all of these references are hereby incorporated herein,
in their
totalities, by reference). Those skilled in the art realize that any nucleic
acid can be
expressed in eukaryotic cells from the appropriate DNA/RNA vector. The
activity of such
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42
nucleic acids can be augmented by their release from the primary transcript by
a ribozyme
(Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa
et al.,
1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids
Res., 19, 5125-
30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al.,
1994, J. Biol.
Chem., 269, 25856; all of these references are hereby incorporated in their
totality by
reference herein).
In another aspect of the invention, RNA molecules of the present invention are
preferably expressed from transcription units (see, for example, Couture et
al., 1996, TIG.,
12, 510) inserted into DNA or RNA vectors. The recombinant vectors are
preferably DNA
plasmids or viral vectors. Ribozyme expressing viral vectors could be
constructed based
on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or
alphavirus.
Preferably, the recombinant vectors capable of expressing the nucleic acid
molecules are
delivered as described above, and persist in target cells. Alternatively,
viral vectors may
be used that provide for transient expression of nucleic acid molecules. Such
vectors
might be repeatedly administered as necessary. Once expressed, the nucleic
acid molecule
binds to the target mRNA. Delivery of nucleic acid molecule expressing vectors
could be
systemic, such as by intravenous or intra-muscular administration, by
administration to
target cells ex-planted from the patient followed by reintroduction into the
patient, or by
any other means that would allow for introduction into the desired target cell
(for a review
see Couture et al., 1996, TIG., 12, 510).
In one aspect, the invention features an expression vector comprising a
nucleic acid
sequence encoding at least one of the nucleic acid molecules of the instant
invention is
disclosed. The nucleic acid sequence encoding the nucleic acid molecule of the
instant
invention is operably linked in a manner which allows expression of that
nucleic acid
molecule.
In another aspect the invention features an expression vector comprising: a) a
transcription initiation region (e.g., eukaryotic pol I, II or III initiation
region); b) a
transcription termination region (e.g., eukaryotic pol I, II or III
termination region); c) a
nucleic acid sequence encoding at least one of the nucleic acid catalyst of
the instant
invention; and wherein said sequence is operably linked to said initiation
region and said
termination region, in a manner which allows expression and/or delivery of
said nucleic
acid molecule. The vector may optionally include an open reading frame (ORF)
for a
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43
protein operably linked on the 5' side or the 3'-side of the sequence encoding
the nucleic
acid catalyst of the invention; and/or an intron (intervening sequences).
Transcription of the nucleic acid molecule sequences are driven from a
promoter for
eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA
polymerase III
(pol 111). Transcripts from pol II or pol III promoters will be expressed at
high levels in all
cells; the levels of a given pol II promoter in a given cell type will depend
on the nature of
the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
Prokaryotic
RNA polymerase promoters are also used, providing that the prokaryotic RNA
polymerase
enzyme is expressed in the appropriate cells (Ekoy-Stein and Moss, 1990, Proc.
Natl.
Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-
72;
Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol.
Cell. Biol., 10,
4529-37). All of these references are incorporated by reference herein.
Several
investigators have demonstrated that nucleic acid molecules, such as ribozymes
expressed
from such promoters can function in mammalian cells (e.g. Kashani-Sabet et
al., 1992,
Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. U S
A, 89,
10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993,
Proc. Natl.
Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO.I., 11, 4411-8;
Lisziewicz et
al., 1993, Proc. Natl. Acad. Sci. U. S. A, 90, 8000-4; Thompson et al., 1995,
Nucleic
Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More
specifically,
transcription units such as the ones derived from genes encoding U6 small
nuclear
(snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating
high
concentrations of desired RNA molecules such as ribozymes in cells (Thompson
et al.,
supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic
Acid Res.,
22, 2830; Noonberg et al., US Patent No. 5,624,803; Good et al., 1997, Gene
Ther., 4, 45;
Beigelman et al., International PCT Publication No. WO 96/18736; all of these
publications are incorporated by reference herein. The above ribozyme
transcription units
can be incorporated into a variety of vectors for introduction into mammalian
cells,
including but not restricted to, plasmid DNA vectors, viral DNA vectors (such
as
adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as
retroviral or
alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).
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44
In yet another aspect, the invention features an expression vector comprising
nucleic
acid sequence encoding at least one of the nucleic acid molecules of the
invention, in a
manner which allows expression of that nucleic acid molecule. The expression
vector
comprises in one embodiment; a) a transcription initiation region; b) a
transcription
termination region; c) a nucleic acid sequence encoding at least one said
nucleic acid
molecule; and wherein said sequence is operably linked to said initiation
region and said
termination region, in a manner which allows expression and/or delivery of
said nucleic
acid molecule. In another preferred embodiment the expression vector
comprises: a) a
transcription initiation region; b) a transcription termination region; c) an
open reading
frame; d) a nucleic acid sequence encoding at least one said nucleic acid
molecule,
wherein said sequence is operably linked to the 3'-end of said open reading
frame; and
wherein said sequence is operably linked to said initiation region, said open
reading frame
and said termination region, in a manner which allows expression and/or
delivery of said
nucleic acid molecule. In yet another embodiment, the expression vector
comprises: a) a
transcription initiation region; b) a transcription termination region; c) an
intron; d) a
nucleic acid sequence encoding at least one said nucleic acid molecule; and
wherein said
sequence is operably linked to said initiation region, said intron and said
termination
region, in a manner which allows expression and/or delivery of said nucleic
acid molecule.
In another embodiment, the expression vector comprises: a) a transcription
initiation
region; b) a transcription termination region; c) an intron; d) an open
reading frame; e) a
nucleic acid sequence encoding at least one said nucleic acid molecule,
wherein said
sequence is operably linked to the 3'-end of said open reading frame; and
wherein said
sequence is operably linked to said initiation region, said intron, said open
reading frame
and said termination region, in a manner which allows expression and/or
delivery of said
nucleic acid molecule.
Examples:
The following are non-limiting examples showing the selection, isolation,
synthesis
and activity of nucleic acids of the instant invention.
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Example 1: Telomerase
The ribonucleoprotein enzyme telomerase consists of an RNA template subunit
and
one or more protein subunits including telomerase reverse transcriptase
(TERT), which
function together to direct the synthesis of telomeres. Telomeres exist as non-
nucleosome
5 DNA/protein complexes at the physical ends of eukaryotic chromosomes. These
capping
structures maintain chromosome stability and replicative potential (Zakian, V.
A., 1995,
Science, 270, 1601-1607). Telomere structure is characterized by tandem
repeats of
conserved DNA sequences rich in G-C base pairs. Additional conserved telomere
elements include a terminal 3'-overhang in the G-rich strand and non-histone
structural
10 proteins that are complexed with telomeric DNA in the nucleus. (Blackburn,
"E., 1990,
JBC., 265, 5919-5921.). Observed shortening of telomeres coincides with the
onset of
cellular senescence in most somatic cell lines lacking significant levels of
telomerase.
This finding has had a profound impact on our views concerning the mechanisms
of aging,
age related disease, and cancer.
15 Conventional DNA polymerises are unable to fully replicate the ends of
linear
chromosomes (Watson, J. D., 1972, Nature, 239, 197-201). This inability stems
from the
3' G-rich overhang that is a product of ribonuclease cleavage of the RNA
primer used in
DNA replication. The overhang prevents DNA polymerise replication since the
recessed
C-rich parent strand cannot be used as a template. Telomerase overcomes this
limitation
20 by extending the 3' end of the chromosome using deoxyribonucleotides as
substrates and a
sequence within the telomerase RNA subunit as a template. (Lingner, J., 1995,
Science,
269, 1533-1534). As such, telomerase is considered a reverse transcriptase
that is
responsible for telomere maintenance.
Telomerase was first discovered by in Tetrahymena thermophila in 1985
(Greider,
25 C. W., 1995, Cell, 43, 405-413). The RNA subunits and their respective
genes were later
discovered and characterized in protozoa, budding yeast, and mammals. Genetic
studies
of these genes confirmed the role of telomerase RNA (TR) in determining
telomere
sequence by mutating genes which encode the telomeric RNA (Yu, G. L., 1990,
Nature,
344, 126-132), (Singer, M. S., 1994, Science, 266, 404-409), (Blasco, M. A.,
1995,
30 Science, 269, 1267-1270). These studies showed that telomerase activity
parallels TR
expression in protozoa, yeast and mice. However, the expression of human
telomerase
RNA (hTR) does not correlate well with telomerase activity in mammalian cells.
Many
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46
human tissues express hTR but are devoid of telomerase activity (Feng, J.,
1995, Science,
269, 1236-1241). Knockout mice, in which the mTR gene has been deleted from
germline
cells, have been shown to be viable for at least six generations. Cells from
later
generations of these mice showed chromosomal abnormalities consistent with
telomere
degradation, indicating that mTR is necessary for telomere length maintenance,
but is not
required for embryonic development, oncogenic transformation, or tumor
formation in
mice (Blasco, M. A., 1997, Cell, 91, 25-34).
The first catalytically active subunit of telomerase (p123) was isolated from
Euplotes
aediculatus along with another subunit (p43) and a 66-kD RNA subunit (Linger,
J., 1996,
Proc. Natl. Acad. Sci., 93, 10712-10717). Subsequent studies revealed
telomerase
catalytic subunit homologs from fission yeast (Est2p) and human genes (TRTl).
The
human homolog, TRT1 encoding hTERT, expressed mRNA with a strong correlation
to
telomerase activity in human cells (Nakamura, T. M., 1997, Science, 277, 955-
959).
Reconstitution of telomerase activity with in vitro transcribed and translated
hTERT and
hTR, either co-synthesized or simply mixed, demonstrated that hTERT and hTR
represent
the minimal components of telomerase. Furthermore, transient expression of
hTERT in
normal diploid human cells restored telomerase activity, demonstrating that
hTERT is the
only component necessary to restore telomerase activity in normal human cells
(Weinrich,
S. L., 1997, Nature Genetics, 17, 498-502). The introduction of telomerase
into normal
human cells using hTERT expression via transfection has resulted in the
extension of life
span in these cells. Such findings indicate that telomere loss in the absence
of telomerase
is the "mitotic clock" that controls the replicative potential of a cell prior
to senescence
(Bodnar, A. G., 1998, Science, 279, 349-352).
Expression of telomerase is observed in germ cell and most cancer cell lines.
These
"immortal" cell lines continue to divide without shortening of their telomeres
(Kim, N.
W., 1994, Science, 266, 2011-2015). A model of tumor progression has evolved
from
these findings, suggesting a role for telomerase expression in malignant
transformation.
Successful malignant transformation in human cells was accomplished for the
first time by
ectopic expression of hTERT in combination with two oncogenes, SV40 large-T
and H-
ras. Injection of nude mice with cells expressing these oncogenes and hTERT
resulted in
rapid growth of tumors. These observations indicate that hTERT mediated
telomere
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47
maintenance is essential for the formation of human tumor cells (Hahn, W. C.,
1999,
Nature, 400, 464-468).
Various methods have been developed to assay telomerase activity in vitro. The
most widely used method to characterize telomerase activity is the telomeric
repeat
amplification protocol (TRAP). TRAP utilizes RT-PCR of cellular extracts to
measure
telomerase activity by making the amount of PCR target dependant upon the
biochemical
activity of the enzyme (Kim, N. W., 1997, Nucleic Acids Research, 25, 2595-
2597, which
is incorporated by reference herein).
A method based on Kim is as follows. Briefly, for the telomerase assay, 2ltg
of
protein extract is used. The extract is assayed in 501 of reaction mixture
containing 0.1
~g TS substrate primer (5~-AATCCGTCGAGCAGAGTT-3', end-labeled using alpha-32P-
ATP and T4 polynucleotide kinase), 0.1 wg ACX return primer(5'-GCGCGG[CTTACC]3
CTAACC-3'), 0.1 ~g NT internal control primer (5'-ATCGCTTCTCGGCCTTTT-3'), 0.01
micromol TSNT internal control template (5'-
AATCCGTCGAGCAGAGTTAAAAGGCCGAGAACGAT-3~), 50 pM each
deoxynucleoside triphosphate, 2 U of Taq DNA polymerase, and 2 w1 CHAPS
protein
extract, all in 1X TRAP buffer (20 mM Tris (pH 8.3), 68 mM KCI, 1.5 mM MgCl2,
1 mM
EGTA, 0.05% Tween 20). Each reaction is placed in a thermocycler block
preheated to 30
C and incubated at 30 C for 10 minutes, then cycled for 27 cycles of 94
degrees C for 30
seconds, 60 degrees C for 30 seconds. Reaction products are separated on a
denaturing 8%
polyacrylamide gel, followed by drying of the gel and autoradiography. The
internal
control (to control for possible Taq polymerase inhibition) generates a band
of 36 nt.
Comparison of radioactive signal integrated (e.g., by phorphorimager analysis)
for
telomerase-extended bands with the radioactive signal from a reaction
performed with a
known amount of quantification standard template (termed R8; 5'-
AATCCGTCGAGCAGAGTTAG [GGTTAG]~-3~) allows expression of telomerase
activity as an absolute value. The absolute value = TPG (total product
generated) =[(TP-
TPi)/TI]/[(R8-B)/RI)] x 100, where TP = telomerase products from test extract,
TPi =
telomerase products from a heat-inactivated (75 C, 10 minutes) extract
reaction, TI = the
signal from the internal control, R8 = the signal from the R8 qualification
standard
template reaction, B = signal from a lysis buffer-only blank reaction, and RI
= the internal
control value for the reaction containing R8 template and NT and TSNT control
primers.
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48
TPG values of 0-10,000 are possible, with the linear range being from
approximately 1 to
1000 TPG. The range of 1 to 1000 TPG encompasses the minimum and maximum
levels
of telomerase activity in most tumor samples tested, while non-tumor cells
most often
have no telomerase activity (TPG approximately zero).
Telomerase activity may also be assayed as follows. Samples to be assayed for
telomerase activity are prepared by extraction into CHAPS lysis buffer (lOmM
Tris pH
7.5, 1mM MgCl2, 1mM EGTA, 0.1 mM PMSF, SmM -mercaptoethanol, 1mM DTT,
0.5% 3-[(3-cholamidopropyl)-dimethyl-amino]-1- propanesulfonate (CHAPS), 10%
glycerol and 40 U/ml RNAse inhibitor (Promega, Madison, WI, U.S.A.). Cells are
suspended in CHAPS lysis buffer and incubated on ice for 30 minutes, which
allows lysis
of 90-100% of cells. Lysate is then transferred to polyallomer centrifuge
tubes and spun at
100,000 x g for 1 hour at 4 degrees C. The supernatant is the protein extract,
and
concentration ranges of 4-10 ~g/~l are suitable for telomerase assay. Extracts
may be
concentrated if necessary using a Microcon Microfilter 30 (Amicron, Beverly,
MA U.S.A.)
according to the manufactureris instructions. Extracts may be stored frozen at
-80 degrees
C until assayed.
A variety of animal models have been designed to assay telomerase activity in
vivo.
Inhibition of telomerase activity has been analyzed in rats via cell
proliferation studies
with MNU (N-methyl-N-nitosurea) induced mammary carcinomas in response to
treatment
with 4-(hydroxyphenyl)retinamide (4-HPR), a known inhibitor of mammary
carcinogenesis in animal models and premenopausal women (Bednarek, A., 1999,
Carcinogenesis, 20, 879-883). Additional studies have focused on the up-
regulation of
telomerase in transformed cell lines from animal and human model systems
(Zhang, P. B.,
1998, Leuk. Res., 22, 509-516), (Chadeneau, C., 1995, Oncogene, 11, 893-898),
(Greenberg, R., 1999, Oncogene, 18, 1219-1226).
Human cell culture studies have been established to assay inhibition of
telomerase
activity in human carcinomas responding to various therapeutics. A human
breast cancer
model for studying telomerase inhibitors is described (Raymond, E., 1999, Br.
J. Cancer,
80, 1332-1341). Human studies of telomerase expression as related to various
other
cancers are described including cervical cancer (Nakano, K., 1998, Am. J.
Pathol, 153,
857-864), endometrial cancer (Kyo, S., 1999, Int. J. Cancer, 80, 60-63),
meningeal
carcinoma (Kleinschmidt-DeMasters, B. K., 1998, J. Neurol. Sci., 161, 124-
134), lung
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49
carcinoma (Yashima, K., 1997, Cancer Reseach, 57, 2372-2377), testicular
cancer in
response to cisplatin (Burger, A. M., 1997, Eur. J. Cancer, 33, 638-644), and
ovarian .
carcinoma (Counter, C. M., 1994, Proc. Natl. Acad. Sci., 91, 2900-2904).
Particular degenerative and disease states that can be associated with
telomerase
expression modulation include but are not limited to:
Cancer: Almost all human tumors have detectable telomerase activity (Shay, J.
W., 1997,
Eur. J. Cancer, 33, 787-791). Treatment with telomerase inhibitors may provide
effective
cancer therapy with minimal side effects in normal somatic cells that lack
telomerase
activity. The therapeutic potential exists for the treatment of a wide variety
of cancer
types.
Restinosis: Telomerase inhibition in vascular smooth muscle cells may inhibit
restinosis by limiting proliferation of these cells.
Infectious disease: Telomerase inhibition in infectious cell types that
express
telomerase activity may provide selective anti-infectious agent activity. Such
treatment
may prove especially effective in protozoan-based infection such as Giardia
and Lesh
Meniesis.
Transplant re'ec~ tion: Telomerase inhibition in endothelial cell types may
demonstrate selective immunnosuppressant activity. Activation of telomerase in
transplant cells could benefit grafting success through increased
proliferative potential.
Autoimmune disease: Telomerase modulation in various immune cells may prove
beneficial in treating diseases such as multiple sclerosis, lupus, and AIDS.
Age related disease: Activation of telomerase expression in cells at or
nearing
senescence as a result of advanced age or premature aging could benefit
conditions such as
macular degeneration, skin ulceration, and rheumatoid arthritis.
The present body of knowledge in telomerase research indicates the need for
methods to assay telomerase activity and for compounds that can regulate
telomerase
expression for research, diagnostic, trait alteration, animal health and
therapeutic use.
Gemcytabine and cyclophosphamide are non-limiting examples of chemotherapeutic
agents that can be combined with or used in conjunction with the nucleic acid
molecules
(e.g. ribozymes and antisense molecules) of the instant invention. Those
skilled in the art
will recognize that other drugs such as anti-cancer compounds and therapies
can be
similarly be readily combined with the nucleic acid molecules of the instant
invention
CA 02403243 2002-02-21
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(e.g. ribozymes and antisense molecules) and are hence within the scope of the
instant
invention. Such compounds and therapies are well known in the art (see for
example
Cancer: Principles and Pranctice of Oncology, Volumes 1 and 2, eds Devita,
V.T.,
Hellman, S., and Rosenberg, S.A., J.B. Lippincott Company, Philadelphia, USA;
5 incorporated herein by reference) and include, without limitations,
antifolates;
fluoropyrimidines; cytarabine; purine analogs; adenosine analogs; amsacrine;
topoisomerase I inhibitors; anthrapyrazoles; retinoids; antibiotics such as
bleomycin,
anthacyclins, mitomycin C, dactinomycin, and mithramycin; hexamethylmelamine;
dacarbazine; 1-asperginase; platinum analogs; alkylating agents such as
nitrogen mustard,
10 melphalan, chlorambucil, busulfan, ifosfamide, 4-
hydroperoxycyclophosphamide,
nitrosoureas, thiotepa; plant derived compounds such as vinca alkaloids,
epipodophyllotoxins, taxol; Tomaxifen; radiation therapy; surgery; nutritional
supplements; gene therapy; radiotherapy such as 3D-CRT; immunotoxin therapy
such as
ricin, monoclonal antibodies herceptin; and the like. For combination therapy,
the nucleic
15 acids of the invention are prepared in one of two ways. First, the agents
are physically
combined in a preparation of nucleic acid and chemotherapeutic agent, such as
a mixture
of a nucleic acid of the invention encapsulated in liposomes and ifosfamide in
a solution
for intravenous administration, wherein both agents are present in a
therapeutically
effective concentration (e.g., ifosfamide in solution to deliver 1000-1250
mg/mz/day and
20 liposome-associated nucleic acid of the invention in the same solution to
deliver 0.1-100
mg/kg/day). Alternatively, the agents are administered separately but
simultaneously in
their respective effective doses (e.g., 1000-1250 mg/m2/d ifosfamide and 0.1
to 100
mg/kg/day nucleic acid of the invention).
Gaeta et al., US patents No. 5,760,062; 5,767,278; 5,770,613 have described
small
25 molecule inhibitors of human telomerase RNA (hTR) subunit.
Blasco et al., 1995, Science, 269, 1267-1270 describe the synthesis and
testing of
antisense oligonucleotides targeted against a specific region of the mouse
telomerase RNA
(mTR) subunit and reported reduction in telomerase activity in mice.
Bisoffi et al., 1998, Eur. J. Cancer, 34, 1242-1249 have studied the down
30 regulation of human telomerase activity by a retrovirus vector expressing
antisense RNA
targeted against the hTR RNA.
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51
Norton et al., 1996, Nature Biotechnology, 14, 615-619 have reported the use
of a
peptide nucleic acid (PNA) molecule targeting hTR RNA to down regulate
telomerase
activity in human immortal breast epithelial cells.
Yokoyama et al., 1998, Cancer Research, 58, 5406-5410 have reported the
synthesis and testing of hammerhead ribozyme constructs targeting hTR RNA
resulting in
a decrease in the telomerase activity in Ishikawa cells.
Henderson, European Patent Application No. 666,313-A2 describes methods of
identifying and cloning hTR gene for use in gene therapy approaches for
creating aberrant
telomeric sequences in transfected human tumor cells. A ribozyme based gene
therapy
approach to inhibit the expression of hTR gene is described as well. The
intended result
of such therapies involves incurred genetic instability based on non-native
telomeric
sequences resulting in rapid cell death of the treated cells.
West et al., US patent No. 5,489,508 describe methods for determining telomere
length and telomerase activity in cells. Inhibitors of hTR RNA, including
oligonucleotides
and/or small molecules are described.
These foregoing approaches of targeting the telomerase RNA subunit (TR) may
not
be very beneficial, because as demonstrated by Feng et al., (Feng, J., 1995,
Science, 269,
1236-1241), telomerase activity in humans does not correlate well to hTR
concentration.
Collins et al., International PCT publication No. WO 98/01542 describes assays
for
the detection of telomerase activity. Four human telomerase subunit proteins
are described
called p140, p105, p48 and p43. In addition, hybridization probes and primers
are
described as inhibitors of telomerase gene function. Antibody based inhibitors
of
telomerase protein subunits are described.
A more attractive approach to telomerase regulation would involve the
regulation of
human telomerase by modulating the expression of the protein subunits of the
enzyme,
preferably the reverse transcriptase (hTERT) subunit. Based of reconstitution
experiments, hTERT and hTR represent the minimal components of telomerase.
Since
hTR expression does not correlate well with telomerase activity in human cells
and since
many human cells express hTR without telomerase activity, targeting hTERT may
prove
more beneficial than targeting hTR. hTERT is the only component necessary to
restore
telomerase activity in normal human cells. A study in which the three major
subunits of
telomerase (hTR, TP1, and hTERT were assayed in normal and malignant
endometrial
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52
tissues determined that hTERT is a rate limiting determinant of enzymatic
activity of
human telomerase (Kyo, S., 1999, Int. J. Cancer, 80, 60-63). Additional
protein subunits
that have been isolated most likely serve only a structural role in telomerase
activity, but
may be important in enhancing the activity of the telomerase enzyme. As such,
hTERT is
one of the better targets for the ectopic regulation of telomerase activity.
Cech et al., International PCT publication No. WO 98/14593 describe
compositions and methods related to hTERT for diagnosis, prognosis and
treatment of
human diseases, for altering proliferative capacity in cells and organisms,
and for
screening compounds and treatments with potential use as human therapeutics.
Cech et al., International PCT publication No. WO 98/14592 describe nucleic
acid
and amino acid sequences encoding various telomerase protein subunits and
motifs of
Euplotes aediculatus, and related sequences from Schizosaccharomyces,
Saccharomyces
sequences, and human telomerase. The polypeptides comprising telomeric
subunits and
functional polypeptides and ribonucleoproteins that contain these subunits are
described as
well. Cech et al., International PCT Publication No. WO 98/14592, mentions in
general
terms the the possibility of using antisense and ribozymes to down regulate
the expression
of human telomerase reverse transcriptase enzyme.
Identification of Potential Target Sites in Human TERT RNA
The sequence of human TERT was screened for accessible sites using a computer
folding algorithm. Regions of the RNA that did not form secondary folding
structures and
contained potential ribozyme and/or antisense binding/cleavage sites were
identified. The
sequences of these cleavage sites are shown in Tables 13-17.
Selection of Enzymatic Nucleic Acid Cleavage Sites in Human TERT RNA
To test whether the sites predicted by the computer-based RNA folding
algorithm
corresponded to accessible sites in TERT RNA, 10 hammerhead ribozyme and three
G-
Cleaver ribozyme sites were selected for further analysis (Table 17). Ribozyme
target
sites were chosen by analyzing sequences of Human TERT (Nakamura et al., 1997
Science 277, 955-959; Genbank sequence accession number: I~1M-003219) and
prioritizing the sites on the basis of folding. Ribozymes were designed that
could bind
each target and were individually analyzed by computer folding (Christoffersen
et al.,
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53
1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al., 1989, Proc. Natl. Acad.
Sci. USA,
86, 7706) to assess whether the ribozyme sequences fold into the appropriate
secondary
structure. Those ribozymes with unfavorable intramolecular interactions
between the'
binding arms and the catalytic core were eliminated from consideration. As
noted below,
varying binding arm lengths can be chosen to optimize activity. Generally, at
least S bases
on each arm are able to bind to, or otherwise interact with, the target RNA.
Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of
TERT RNA
Ribozymes were designed to anneal to various sites in the RNA message. The
binding arms are complementary to the target site sequences described above.
The
ribozymes were chemically synthesized. The method of synthesis used followed
the
procedure for normal RNA synthesis as described above and in Usman et al.,
(1987 J.
Am. Chem. Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18,
5433) and
Wincott et al., supra, and made use of common nucleic acid protecting and
coupling
groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-
end. The
average stepwise coupling yields were >98%.
Ribozymes were also synthesized from DNA templates using bacteriophage T7
RNA polyrnerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51).
Ribozymes
were purified by gel electrophoresis using general methods or were purified by
high
pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality
of which is
hereby incorporated herein by reference) and were resuspended in water. The
sequences of
the chemically synthesized ribozymes used in this study are shown below in
Table 13-17.
Ribozyme Cleavage of TERT RNA Target in vitro
Ribozymes targeted to the human TERT RNA are designed and synthesized as
described above. These ribozymes can be tested for cleavage activity in vitro,
for example
using the following procedure. The target sequences and the nucleotide
location within the
TERT RNA are given in Tables 13-17.
Cleavage Reactions: Full-length or partially full-length, internally-labeled
target
RNA for ribozyme cleavage assay is prepared by in vitro transcription in the
presence of
[a-32p] CTP, passed over a G 50 Sephadex column by spin chromatography and
used as
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54
substrate RNA without further purification. Alternately, substrates are 5'-32P-
end labeled
using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming 15
~1 of a
2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-
HCI, pH
7.5 at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by
adding the 2X
ribozyme mix to an equal volume (15 ~l) of substrate RNA (maximum of 1-5 nM; 5
x 105
to 1 x 10' cpm) that was also pre-warmed in cleavage buffer. As an initial
screen, assays
are carried out for 1 hour at 37°C using a final concentration of
either 40 nM or 1 mM
ribozyme, i.e., ribozyme excess. The reaction is quenched by the addition of
an equal
volume (30 w1) of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05%
xylene cyanol after which the sample is heated to 95°C for 2 minutes,
quick chilled and
loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific
RNA
cleavage products generated by. ribozyme cleavage are visualized on an
autoradiograph of
the gel. The percentage of cleavage is determined by Phosphor Imager~
quantitation of
bands representing the intact substrate and the cleavage products.
Example 2: PTP-1B
Protein tyrosine phosphorylation and dephosphorylation are important
mechanisms
in the regulation of signal transduction pathways that control the processes
of cell growth,
proliferation, and differentiation (Fantl, W. J., 1993, Annu. Rev. Biochem.,
62, 453-481).
Cooperative enzyme classes regulate protein tyrosine phosphorylation and
dephosphorylation events. These broad classes of enzymes consist of the
protein tyrosine
kinases (PTKs) and protein tyrosine phosphatases (PTPs). PTKs and PTPs can
exist as
both receptor-type transmembrane proteins and as cytoplasmic protein enzymes.
Receptor
tyrosine kinases propagate signal transduction events via extracellular
receptor-ligand
interactions that result in the activation of the tyrosine kinase portion of
the PTK in the
cytoplasmic domain. Receptor-like transmembrane PTPs function through
extracellular
ligand binding that modulates dephosphorylation of intracellular
phosphotyrosine proteins
via cytoplasmic phosphatase domains. Cytoplasmic PTKs and PTPs exert enzymatic
activity without receptor-mediated ligand interactions, however,
phosphorylation can
regulate the activity of these enzymes.
CA 02403243 2002-02-21
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Protein tyrosine phosphatase 1B, a cytoplasmic PTP, was the first PTP to be
isolated
in homogeneous form (Tonks, N. K., 1988, J. Biol. Chem., 263, 6722-6730),
characterized
(Tonks, N. K., 1988, J. Biol. Chem., 263, 6731-6737), and sequenced
(Charbonneau, H.,
1989, Biochemistry, 86, 5252-5256). Cytoplasmic and receptor-like PTPs both
share a
5 catalytic domain characterized by eleven conserved amino acids containing
cysteine and
arginine residues that are critical for phosphatase activity (Streuli, M.,
1990, EMBO, 9,
2399-2407). A cysteine residue at position 215 is responsible for the covalent
attachment
of phosphate to the enzyme (Guar, K., 1991, J. Biol. Chem., 266, 17026-17030).
The
crystal structure of human PTP1B defined the phosphate binding site of the
enzyme as a
10 glycine rich cleft at the surface of the molecule with cysteine 215
positioned at the base of
this cleft. The location of cysteine 215 and the shape of the cleft provide
specificity of
PTPase activity for tyrosine residues but not for serine or threonine residues
(Barford, D.,
1994, Science, 263, 1397-1404).
Receptor tyrosine kinase and protein tyrosine phosphatase localization plays a
key
15 role in the regulation of phosphotyrosine mediated signal transduction. PTP-
1B activity
and specificity against a panel of receptor tyrosine kinases demonstrated
clear differences
between substrates, suggesting that cellular compartmentalization is a
determinant in
defining the activity and function of the enzyme (Lammers, 8.,1993, J. Biol.
Chem., 268,
22456-22462). Experiments have indicated that PTP-1B is localized
predominantly in the
20 endoplasmic reticulum via its 35 amino acid carboxyterminal sequence. PTP-
1B is also
tightly associated with microsomal membranes with its catalytic phosphatase
domain
oriented towards the cytoplasm (Frangioni, J. V., 1992, Cell, 68, 545-560).
PTP-1B has been identified as a negative regulator of the insulin response.
PTP-1B
is widely expressed in insulin sensitive tissues (Goldstein, B. J., 1993,
Receptor, 3, 1-15).
25 Isolated PTP-1B dephosphorylates the insulin receptor in vitro (Tonks, N.
K., 1988, J.
Biol. Chem., 263, 6731-6737). PTP-1B dephosphorylation of multiple
phosphotyrosine
residues of the insulin receptor proceeds sequentially and with specificity
for the three
tyrosine residues that are critical for receptor autoactivation (Ramachandran,
C., 1992,
Biochemistry, 31, 4232-4238). In addition to insulin receptor
dephosphorylation, PTP-1B
30 also dephosphorylates the insulin related subtrate 1 (IRS-1), a principal
substrate of the
insulin receptor (Lammers, R., 1993, J. Biol. Chem., 268, 22456-22462).
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Microinjection of PTP1B into Xenopus oocytes results in the inhibition of
insulin
stimulated tyrosine phosphorylation of endogenous proteins, including the ~3-
subunit of the
insulin and insulin-like growth factor receptor proteins. The resulting 3 to 5
fold increase
over endogenous PTPase activity also blocks the activation of an S6 peptide
kinase
(Cicirelli, M. F., 1990, Proc, Natl. Acad. Sci., 87, 5514-5518). Inactivation
of
recombinant rat PTP-1B with antibody immunoprecipitation results in the
dramatic
increase in insulin stimulated DNA synthesis and phosphatidylinositol 3'-
kinase activity.
Insulin stimulated receptor autophosphorylation and insulin receptor substrate
1 tyrosine
phosphorylation are increased dramatically as well through PTP-1B inhibition
(Ahmad, F.,
1995, J. Biol. Chem., 270, 20503-20508).
Increased PTP-1B expression correlates with insulin resistance in
hyperglycemic
cultured fibroblasts. In this study, desensitized insulin receptor function
was observed via
impaired insulin-induced autophosphorylation of the receptor. Treatment with
insulin
sensitivity normalizing thiazolidine derivatives resulted in the amelioration
of the
hyperglycemic insulin resistance via a normalization in PTP-1B expression
(Maegawa, H.,
1995, J. Biol. Chem., 270, 7724-7730). A marine model of insulin resistance
with a
knockout of the hetrerotrimeric GTP-binding protein subunit Gia2 provides a
type 2
diabetis phenotype that correlates with the increased expression of PTP-1B
(Moxam, C.
M., 1996, Nature, 379, 840-844).
PTP-1B interacts directly with the activated insulin receptor ~i-subunit. An
inactive
homolog of PTP-1B was used to precipitate the activated insulin receptor in
both purified
receptor preparations and whole-cell lysates. Phosphorylation of the insulin
receptor's
triple tyrosine residues in the kinase domain is necessary for PTP-1B
interaction.
Furthermore, insulin stimulates tyrosine phosphorylation of PTP-1B (Seely, B.
L., 1996,
Diabetes, 45, 1379-1385). A similar study confirmed the direct interaction of
PTP-1B
with the insulin receptor (3-subunit as well as the required multiple
phosphorylation sites
within the receptor and PTP-1B (Bandyopadhyay, D., J. Biol. Chem., 272, 1639-
1645).
Knockout mice lacking the PTP-1B gene (both homozygous PTP-1B-/- and
heterozygous PTP-1B+/-) have been used to study the specific role of PTP-1B
relating to
insulin action in vivo. The resulting PTP-1B deficient mice were healthy and,
in the fed
state, had lower blood glucose and circulating insulin levels that were half
that of their
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PTP-1B+/+ expressing littermates. These PTP-1B deficient mice demonstrated
enhanced
insulin sensitivity in glucose and insulin tolerance tests. At the
physiological level, the
PTP-1B deficient mice showed increased phosphorylation of the insulin receptor
after
insulin administration. When fed a high fat diet, the PTP-1B deficient mice
were resistant
to weight gain and remained insulin sensitive as opposed to normal PTP-1B
expressing
mice, who rapidly gained weight and become insulin resistant (Elchebly, M.,
1999,
Science, 283, 1544-1548). As such, modulation of PTP-1B expression could be
used to
regulate autophosphorylation of the insulin receptor and increase insulin
sensitivity in vivo.
This modulation could prove beneficial in the treatment of insulin related
disease states.
In light of the above findings, particular disease states that involve PTP-1B
expression include but are not limited to:
Diabetes: Both type 1 and type 2 diabetes may be treated by modulation of PTP-
1B
expression. Type 2 diabetes correlates to desensitized insulin receptor
function (White et
al., 1994). Disruption of the PTP-1B dephosphorylation of the insulin receptor
in vivo
manifests in insulin sensitivity and increased insulin receptor
autophosphorylation
(Elchebly et al., 1999). Insulin dependant diabetes, type 1, may respond to
PTP-1B
modulation through increased insulin sensitivity.
Obesity: Elchebly et al., 1999, demonstrated that PTP-1B deficient mice were
resistant to
weight gain when fed a high fat diet compared to normal PTP-1B expressing
mice. This
finding suggests that PTP-1B modulation may be beneficial in the treatment of
obesity.
Ahmad et al., 1997, Metab. Clin. Exp., 46, 1140-1145, describe reduced PTPs in
adipose
tissue and improved insulin sensitivity in obese subjects following weight
loss.
Troglitazone is a non-limiting example of a pharmaceutical agent that can be
combined with or used in conjunction with the nucleic acid molecules (e.g.
ribozymes and
antisense molecules) of the instant invention. Those skilled in the art will
recognize that
other drugs such as anti-diabetes and anti-obesity compounds and therapies can
be
similarly be readily combined with the nucleic acid molecules of the instant
invention
(e.g. ribozymes and antisense molecules) are hence within the scope of the
instant
invention.
Methods have been developed to assay PTP-1B activity.
Maegawa et al., 1995, J. Biol. Chem., 270, 7724-7730, describe a tissue
culture
model in which Rat 1 fibroblasts expressing human insulin receptors can be
used to model
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hyperglycemia induced insulin resistance. Maegawa et al. also describe assays
to measure
PTPase activity using labeled phosphorylated insulin receptors and by
immunoenzymatic
techniques.
Moxham et al., 1996, Nature, 379, 840-844, describe a marine animal and tissue
culture model employing Gia2 deficiency to study hyperinsulinaemia, impaired
glucose
tolerance and resistance to insulin in vivo. Assays for PTPase activity and
tyrosine
phosphorylation of insulin-receptor substrate 1 are described.
Khandelwal et al., 1995, Molecular and Cellular Biochemistry, 153, 87-94,
describe
four different animal models for studying insulin dependent and insulin
resistant diabetes
mellitus. These models were used to study the effect of vanadate, an insulin
mimetic and
PTPase inhibitor, on the insulin-stimulated phosphorylation of the insulin
receptor and its
tyrosine kinase acitivity.
Wang et al., 1999, Biochim. Biophys. Acta, 1431, 14-23, describe fluorescein
monophosphates as fluorogenic substrates for PTPs.
Various methods and compounds have been developed to inhibit protein tyrosine
phosphatase activity.
Wrobel et al., 1999, J. Med. Chem., 42, 3199-3202, describe PTP-1B inhibition
and
antihyperglycemic activity in the ob/ob mouse model by 11-
arylbenzo[b]naphtho[2,3-
d]furans and arylbenzo[b]naphtho[2,3-d]thiophenes.
Andersen et al., International PCT publication No. WO 98/DK407 describe the
preparation of thienopyridzinones and thienochromenones as modulators of
PTPases.
Taing et al., 1999, Biochemistry, 38, 3793-3803, describe potent and highly
selective
inhibitors of PTP-1B comprising an array of bis(aryldifluorophosphonates).
Ham et al., 1999, Bioorg. Med. Chem. Lett., 9, 185-186, describe selective
inactivation of PTP-1B by a sulfone analog of naphthoquinone.
Desmarais et al., 1999, Biochem, J., 337, 219-223, describe
[Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of PTPs.
Taylor et al., 1998, Bioorg. Med. Chem., 6, 2235, describe potent non-peptidyl
inhibitors of PTP-1B.
Kotoris et al., 1998, Bioorg. Med. Chem. Lett., 8, 3275-3280, describe novel
phosphate mimetics for the design of non-peptidyl inhibitors of PTPs.
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Groves et al., 1998, Biochemistry, 37, 17773-17783, describe the structural
basis for
PTP-1B inhibition by the phosphotyrosine peptide mimetics
(difluoronaphthylmethyl)phosphoric acid and the fluoromalonyl tyrosines with
complexed
crystal structures.
Yao et al., 1998, Bioorgl Med. Chem., 6, 1799-1810, describe the structure-
based
design and synthesis of small molecule PTP-1B inhibitors comprising novel
naphthyldifluoromethyl phosphoric acids 1 and 2.
Taylor et al., 1998, Bioorg. Med. Chem., 6, 1457-1468, describe potent non-
peptidyl
inhibitors of PTP-1B.
Desmarais et al., 1998, Arch. Biochem. Biophys., 354, 225-231, describe
inhibition
of PTP-1B and CD45 by sulfotyrosyl peptides.
Mjalli et al., application US 96-766114, cont. in part of US patent No.
543,630,
describe the preparation of heterocyclic compounds as modulators of proteins
with
phosphotyrosine recognition units.
Wang et al., 1998, Bioorg. Med. Chem. Lett., 8, 345-350, describe
naphthalenebis[a,a-difluoromethylenephosphonates] as potent inhibitors of
PTPs.
Rice et al., 1997, Biochemistry, 36, 15965-15974, describe a targeted library
of
small molecule tyrosine and dual-specificity phosphatase inhibitors with
random side
chain variation from a rational core design.
Olefsky, International PCT publication No. WO 97/LJS2752 describes a method
and
phosphopeptides used for the treatment of insulin resistance based on the
association of
PTP-1B with the activated insulin receptor. Also included is a method for
determining
whether a compound inhibits PTP-1B binding to the insulin receptor.
Huyer et al., 1997, J. Biol. Chem., 272, 843-851, describe the mechanism of
inhibition of PTPases by vanadate and pervanadate.
Burke et al., 1996, Biochemistry, 35, 15989-15996, describe the structure-
based
design of PTP-1B inhibitors.
Tonks et al., International PCT publication No. WO 97/U5 13016, describe
substrate-trapping protein PTPase mutants for identification of tyrosine-
phosphorylated
protein substrates and their clinical uses.
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The human genome is thought to contain up to 100 PTPases, each varying
slightly in
chemistry but vastly in function. Compounds designed to inhibit PTP-1B
activity
specifically by covalent binding to or modification of PTP-1B have the
potential for
multiple side effects. Conventional drug substances that will potently
suppress PTP-1B
5 activity with few or no side effects from interaction with other PTPs are
difficult to
envision. A more attractive approach to PTP-1B modulation would involve the
specific
regulation of PTP-1B expression with oligonucleotides.
Identification of Potential Target Sites in Human PTP-1B RNA
10 The sequence of human PTP-1B was screened for accessible sites using a
computer
folding algorithm. Regions of the RNA that did not form secondary folding
structures and
contained potential ribozyme and/or antisense binding/cleavage sites were
identified. The
sequences of these cleavage sites are shown in Tables 3-8.
15 Selection of Enzymatic Nucleic Acid Cleavage Sites in Human PTP-1B RNA
To test whether the sites predicted by the computer-based RNA folding
algorithm
corresponded to accessible sites in PTP-1B RNA, 10 hammerhead ribozyme, five
NCH
and three G-Cleaver ribozyme sites were selected for further analysis (Table
8).
Ribozyme target sites were chosen by analyzing sequences of Human PTP-1B
(Genbank
20 accession number M33689) and prioritizing the sites on the basis of
folding. Ribozymes
were designed that could bind each target and were individually analyzed by
computer
folding (Christoffersen et al., 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger
et al., 1989,
Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences
fold into
the appropriate secondary structure. Those ribozymes with unfavorable
intramolecular
25 interactions between the binding arms and the catalytic core were
eliminated from
consideration. As noted below, varying binding arm lengths can be chosen to
optimize
activity. Generally, at least 5 bases on each arm are able to bind to, or
otherwise interact
with, the target RNA.
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Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of PTP-
1B RNA
Ribozymes were designed to anneal to various sites in the RNA message. The
binding arms are complementary to the target site sequences described above.
The
ribozymes were chemically synthesized. The method of synthesis used followed
the
procedure for normal RNA synthesis as described above and in Usman et al., (
1987 J.
Am. Chem. Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18,
5433) and
Wincott et al., supra, and made use of common nucleic acid protecting and
coupling
groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-
end. The
average stepwise coupling yields were >98%.
Ribozymes were also synthesized from DNA templates using bacteriophage T7
RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51).
Ribozymes
were purified by gel electrophoresis using general methods or were purified by
high
pressure liquid chromatography (HPLC; see Wincott et al., supra; the totality
of which is
hereby incorporated herein by reference) and were resuspended in water. The
sequences of
the chemically synthesized ribozymes used in this study are shown below in
Tables 3-8.
Ribozyme Cleavage of PTP-1B RNA Target in vitro
Ribozymes targeted to the human PTP-1B RNA are designed and synthesized as
described above. These ribozymes can be tested for cleavage activity in vitro,
for
example, using the following procedure. The target sequences and the
nucleotide location
within the PTP-1B RNA are given in Tables 3-8.
Cleavage Reactions: Full-length or partially full-length, internally-labeled
target
RNA for ribozyme cleavage assay is prepared by in vitro transcription in the
presence of
[a-32p] CTP, passed over a G 50 Sephadex column by spin chromatography and
used as
substrate RNA without fiuther purification. Alternately, substrates are 5'-32P-
end labeled
using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a
2X
concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-
HCI, pH 7.5
at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by adding
the 2X
ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was
also
pre-warmed in cleavage buffer. As an initial screen, assays are carried out
for 1 hour at
37°C using a final concentration of either 40 nM or 1 mM ribozyme,
i.e., ribozyme excess.
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The reaction is quenched by the addition of an equal volume of 95% formamide,
20 mM
EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is
heated to 95~C for 2 minutes, quick chilled and loaded onto a denaturing
polyacrylamide
gel. Substrate RNA and the specific RNA cleavage products generated by
ribozyme
cleavage are visualized on an autoradiograph of the gel. The percentage of
cleavage is
determined by Phosphor Imager~ quantitation of bands representing the intact
substrate
and the cleavage products.
Example 3: MetAP-2
Methionyl aminopeptidases are metalloproteases that are known to possess post-
translational enzymatic activity by hydrolytically cleaving amino-terminal
methionine
residues from nascent peptide substrates in a non-processive manner (Kendall,
R. L., 1992,
J. Biol. Chem., 267, 20667-20673). This family of enzymes is divided into two
classes
(type 1 and type 2) based on differences in sequence, although the overall
structure of the
two classes are similar (Liu, S., 1998, Science, 282, 1324-1327). Methionine
aminopeptidase expression appears to be involved in the control of cellular
proliferation.
Deletion of the MetAP gene from E. Coli is lethal (Chang, S. Y., 1989, J.
Bacteriol., 171,
4071-4072). In Saccharomyces cerevisiae, deletion of the gene that codes for
either
MetAP-1 or 2 results in a slow growth phenotype while deletion of both genes
is lethal (Li,
X., 1995, Proc. Natl. Acad. Sci., 92, 12357-12361). (Human methionine
aminopeptidase-
1, MetAP-1, accession No. P53582).
The aminopeptidase function of this class of enzymes may serve a regulatory
role in
activating signal peptides in conjunction with N-myristoyl transferase (NMT)
activity.
NMT is expressed from a lethal gene in yeast (Duronio, R. J., 1989, Science,
243, 796-
800). NMT is responsible for amino-terminal ligation of myristic acid onto
nascent
peptides and cannot act on peptides with an amino-terminal methionine residue
(Resh, M.
D., 1996, Cell. Signal., 8, 403-412). Myristoylation of proteins correlates to
intracellular
localization events that may determine why certain signaling proteins are
dependent on
NMT for activity (Taunton, J., 1997, Chemistry & Biology, 4, 493-496). Protein
tyrosine
kinase Src is dependant on myristoylation for activity and has been identified
as an
upstream regulator of human vascular endothelial growth factor (VEGF)
expression
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63
through hypoxic induction in solid tumors (Mukhopadhyay, D., 1995, Nature,
375, 577-
581). MetAPs may therefore regulate the activation of signal peptides (such as
VEGF)
through cotranslational modification of nascent peptides with NMT. Disruption
of protein
myristoylation by MetAP inhibition could result in the improper localization
of signaling
proteins resulting in inhibition of cell growth. (Human N-
myristoyltransferase, hNMT,
accession No. AF043324.)
Fumagillin, a sesquiterpene diepoxide metabolite of the fungus Aspergillus
fumigates, and a related compound TNP-470, are strong inhibitors of growth in
cultured
endothelial cells. The antiproliferative and angiostatic activity of
fumagillin was originally
discovered by the serendipitous contamination of Aspergillus fumigates in an
endothelial
cell culture dish in which cells closest to the fungal colony displayed growth
inhibition.
Synthetic analogs of fiunagillin were later synthesized resulting in the
discovery of TNP-
470, which is 50 times more potent of an inhibitor than fumagillin and is less
toxic in mice
(Ingber, D., 1990, Nature, 348, 555-557). Treatment of endothelial cells with
these
compounds results in late G1 phase arrest. TNP-470 inhibits the signaling
pathway of
retinoblastoma gene product phosphorylation, cyclin dependent kinases cdk2 and
cdk4
activation, and cyclins E and A expression (Abe, J., 1994, Cancer Res., 54,
3407-3412).
TNP-470 has also been shown to potently inhibit endothelial cell proliferation
induced by
the growth factors VEGF and bFGF (Toi, M., 1994, Oncology Reports, 1, 423-
426).
The bifunctional protein MetAP-2 has been identified as the molecular target
for
fumagillin and related compounds that demonstrate antiproliferative activity
in endothelial
cells. The use of affinity chromatography with a fumagillin-biotin conjugate
resulted in
the isolation of a 67-kDa mammalian protein through covalent interaction with
the bound
substrate. Analysis of digested peptide fragments from the isolated protein
revealed
MetAP-2 as the covalently bound substrate. Subsequent growth inhibition
studies in yeast
utilizing MetAP-1 and MetAP-2 deletion strains determined that MetAP-2 is
selectively
inhibited by fumagillin in vivo (Sin, N., 1997, Proc. Natl. Acad. Sci., 94,
6099-6103). A
similar study with TNP-470 and ovalicin, another potent inhibitor of
neovascularization,
determined that MetAP-2 is the molecular target for these fumagillin-related
compounds
(Griffith, E. C., 1997, Chemistry & Biology, 4, 461-471).
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64
MetAP-2 expression correlates with cellular growth. Non-dividing cells in
culture
have no detectable levels of the 67-kDa MetAP-2 protein by immunoassay. MetAP-
2 has
been shown to affect translational initiation by association with eukaryotic
initiation factor
2a (eIF-2a) (Ray, M. K., 1992, Proc. Natl. Acad. Sci., 89, 539-543). The
binding of
MetAP-2 with eIF-2a inhibits the heme-regulated inhibitor kinase (HRI)
phosphorylation
of eIF-2a in vitro in reticulocyte lysates (Datta, B., 1988, Proc. Natl. Acad.
Sci., 85, 3324-
3328). MetAP-2/eIF-2a binding results in the partial reversal of protein
synthesis
inhibition by double stranded RNA dependent kinase mediated phosphorylation in
vivo
(Wu, S., 1996, Biochemistry, 35, 8275-8280). Griffith et al. also determined
that covalent
binding of TNP-470 and ovalicin, while potently inhibiting methionine
aminopeptidase
type 2 activity specifically, did not affect the regulatory activity of MetAP-
2 on eIF-2a.
This finding by Griffith et al. rules out the possibility that control of eIF-
2a
phosphorylation by MetAP-2 is responsible for the inhibition of endothelial
cell
proliferation by fumagillin related compounds.
Particular angiogenesis related degenerative and disease states that can be
associated
with MetAP expression modulation include but are not limited to:
Cancer: Solid tumors are unable to grow or metastasize without the formation
of new
blood vessels (Hanahan, D., 1996, Cell, 86, 353-364). Inhibition of
angiogenesis via
MetAP modulation can potentially be used to treat a wide variety of cancers.
Diabetic retinopathy and aye related macular degeneration: Ocular
neovascularization is observed in diabetic retinopathy, which is mediated by
up-regulation
of VEGF (Adamis, A. P., 1994, Amer. J. Ophthal., 118, 445-450). The
requirement of
protein kinase Src in hypoxia induced VEGF expression (Mukhopadhyay, D., 1995,
Nature, 375, 577-581) indicates that MetAP modulation of aminopeptidase
activity can
potentially be used to treat conditions involving ocular neovascularization.
Arthritis: The ingrowth of a vascular pannus in arthritis may be mediated by
the
overexpression of angiogenic factors from infiltrating inflammatory cells,
macrophages,
and immune cells (Peacock, D. J., 1992, J. exp. Med., 175, 1135-1138).
Angiogenesis
inhibition through MetAP modulation can potentially be used to treat
arthritis.
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Psoriasis: Angiogenesis has been implicated in psoriasis due to overexpression
of
the angiogenic polypeptide interleukin-8 and decreased expression of the
angiogenesis
inhibitor thrombospondin (Nickoloff, B. J., 1994, Amer. J. Pathol. 44, 820-
828).
Angiogenesis inhibition through MetAP modulation can potentially be used to
treat
5 psoriasis.
Female reproduction: Angiogenesis in the female reproductive system has been
implicated in several disorders of the reproductive tract (Reynolds, L. P.,
1992, FASEB, 6,
886-892). Modulation of angiogenesis through control of MetAP may have various
applications in the area of female reproduction and fertility.
10 Various methods have been developed to assay MetAP activity.
Griffith et al., 1998, Proc. Natl. Acad. Sci., 95, 15183-15188, describe an
enzymatic
assay for MetAP-2 activity in vitro and an endothelial cell culture
proliferation assay for
MetAP-2 activity in vivo.
Weber et al., 1999, International PCT publication No. WO 98/US-21231 describe
15 novel fluorescent reporter molecules and an enzymatic assay that can be
used for
determining the activity of MetAP-2 for drug screening and determining the
chemosensitivity of human cancer cells to treatment with chemotherapeutic
drugs.
Larrabee, J. A. et al., 1999, Anal. Biochem, 269, 194-198, describe the use of
a high-
pressure liquid chromatographic (HPLC) method for assaying MetAP-2 activity
with
20 application to the study of enzymic inactivation.
Quantitative methods have been developed to assay the efficacy of
antiangiogenic
therapies.
Wantanabe et al., 1992, Molec. Biol. Cell, 3, 324a, describe the quantitation
of
angiogenic peptides (bFGF) in human serum as a prognostic test for breast
cancer.
25 Nguyen et al., 1994, J. Natn. Cancer Inst., 86, 356-361, describe the
quantitation of
angiogenic peptides (bFGF) in the urine of patients with a wide spectrum of
cancers.
Li et al., 1994, The Lancet, 344, 82-86, describe the quantitation of
angiogenic
peptides (bFGF) in the cerebrospinal fluid of children with brain tumors. This
work also
describes determining the extent of neovascularization in histological
sections by utilizing
30 microvessel count.
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66
The present body of knowledge in angiogenesis research indicates the need for
compounds that can modulate MetAP activity for research, diagnostic, trait
alteration,
animal health and therapeutic use.
Griffith et al., International PCT publication No. WO 9856372 describe small
molecule inhibitors of MetAP2 and uses thereof.
D'Amato et al., International PCT publication No. WO 9805293 describe the use
of
AGM-1470 (TNP-470) as an angiogenesis inhibitor for use in regulating the
female
reproductive system and for treating diseases of the reproductive tissue.
Davidson et al., US patent No. 5,801,146 describe a compound and method for
inhibiting angiogenesis using mammalian kringle 5 protein.
Cao et al., US patent No. 5,854,221 describe a protein-based endothelial cell
proliferation inhibitor and its method of use.
Chang et al., US patent No. 5,888,796 describe a clone of a nucleotide
sequence
encoding a protein having two functions comprising methionine aminopeptidase
activity
and anti eIF-2 phosphorylation activity.
Wang et al., 1998, Proc. Am. Assoc. Cancer Res., 39, 98 (abstr.) describe
blocked
proliferation of human endothelial cells by human MetAP-2 antisense
oligonucleotides.
A rat corneal model has been developed to study ribozyme inhibition of VEGF
receptor-mediated angiogenesis (Pavco, P. A., 1999, Nucleic Acids Research,
27, 2569-
2577). A similar study employing MetAP-2 inhibition could be used to study
ribozyme
based inhibition of MetAP-2 induced angiogenesis in vivo.
Identification of Potential Target Sites in Human MetAP-2 RNA
The sequence of human MetAP-2 was screened for accessible sites using a
computer-folding algorithm. Regions of the RNA that did not form secondary
folding
structures and contained potential ribozyme and/or antisense binding/cleavage
sites were
identified. The sequences of these cleavage sites are shown in Tables 9-12.
Selection of Enzymatic Nucleic Acid Cleava~~e Sites in Human MetAP-2 RNA
To test whether the sites predicted by the computer-based RNA folding
algorithm
corresponded to accessible sites in MetAP-2 RNA, 11 hammerhead ribozyme, 4 NCH
and
three G-Cleaver ribozyme sites were selected for further analysis (Table 12).
Ribozyme
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67
target sites were chosen by analyzing sequences of Human MetAP-2 (Genbank
accession
number HSU29607) and prioritizing the sites on the basis of folding. Ribozymes
were
designed that could bind each target and were individually analyzed by
computer folding
(Christoffersen et al., 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al.,
1989, Proc.
Natl. Acad. Sci. USA, 86, 7706) to assess whether the ribozyme sequences fold
into the
appropriate secondary structure. Those ribozymes with unfavorable
intramolecular
interactions between the binding arms and the catalytic core were eliminated
from
consideration. As noted below, varying binding arm lengths can be chosen to
optimize
activity. Generally, at least 5 bases on each arm are able to bind to, or
otherwise interact
with, the target RNA.
Chemical Synthesis and Purification of Ribozymes for Efficient Cleavage of
MetAP-2
RNA
Ribozymes were designed to anneal to various sites in the RNA message. The
binding arms are complementary to the target site sequences described above.
The
ribozymes were chemically synthesized. The method of synthesis used followed
the
procedure for normal RNA synthesis as described above and in Usman et al.,
(1987 J.
Am. Chem. Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18,
5433) and
Wincott et al., supra, and made use of common nucleic acid protecting and
coupling
groups, such as dimethoxytrityl at the S'-end, and phosphoramidites at the 3'-
end. The
average stepwise coupling yields were >98%.
Ribozymes were also synthesized from DNA templates using bacteriophage T7
RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, S 1 ).
Ribozymes
were purified by gel electrophoresis using general methods or were purified by
high
pressure liquid chromatography (HPLC; see Wincott et al., supra; the totality
of which is
hereby incorporated herein by reference) and were resuspended in water. The
sequences of
the chemically synthesized ribozymes used in this study are shown below in
Table 9-12.
Riboz~me Cleavage of MetAP-2 RNA Target in vitro
Ribozymes targeted to the human MetAP-2 RNA are designed and synthesized as
described above. These ribozymes can be tested for cleavage activity in vitro,
for
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example, using the following procedure. The target sequences and the
nucleotide location
within the MetAP-2 RNA are given in Tables 9-12.
Cleavage Reactions: Full-length or partially full-length, internally-labeled
target
RNA for ribozyme cleavage assay is prepared by in vitro transcription in the
presence of
[a-32p] CTP, passed over a G 50 Sephadex column by spin chromatography and
used as
substrate RNA without further purification. Alternately, substrates are S'-32P-
end labeled
using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a
2X
concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-
HCI, pH 7.5
at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by adding
the 2X
ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was
also
pre-warmed in cleavage buffer. As an initial screen, assays are carried out
for 1 hour at
37°C using a final concentration of either 40 nM or 1 mM ribozyme,
i.e., ribozyme excess.
The reaction is quenched by the addition of an equal volume of 95% formamide,
20 mM
EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is
heated to 95°C for 2 minutes, quick chilled and loaded onto a
denaturing polyacrylamide
gel. Substrate RNA and the specific RNA cleavage products generated by
ribozyme
cleavage are visualized on an autoradiograph of the gel. The percentage of
cleavage is
determined by Phosphor Imager~ quantitation of bands representing the intact
substrate
and the cleavage products.
Example 4: BACE, ps-1, ps-2
Alzheimer's disease (AD) is a progressive, degenerative disease of the brain
which
affects approximately 4 million people in the United States alone. An
estimated 14
million Americans will have Alzheimer's disease by the middle of the next
century if no
cure or definitive prevention of the disease is found. Nearly one out of ten
people over age
65 and nearly half of those over 85 have Alzheimer's disease. Alzheimer's
disease is not
confined to the elderly, a small percentage of people in their 30's and 40's
are afflicted
with early onset AD. Alzheimer's disease is the most common form of dementia,
and
amounts to the third most expensive disease in the US following heart disease
and cancer.
An estimated 100 billion dollars are spent annually on Alzheimer's disease
(National
Alzheimer's Association, 1999).
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Alzheimer's disease is characterized by the progressive formation of insoluble
plaques and vascular deposits in the brain consisting of the 4 kD amyloid ~3
peptide (A~i).
These plaques are characterized by dystrophic neurites that show profound
synaptic loss,
neurofibrillary tangle formation, and gliosis. A~3 arises from the proteolytic
cleavage of
the large type I transmembrane protein, ~3-amyloid precursor protein (APP)
(Kang et al.,
1987, Nature, 325, 733). Processing of APP to generate A(3 requires two sites
of cleavage
by a (3-secretase and a y-secretase. ~3-secretase cleavage of APP results in
the cytoplasmic
release of a 100 kD soluble amino-terminal fragment, APPs(3, leaving behind a
12 kD
transmembrane carboxy-terminal fragment, C99. Alternately, APP can be cleaved
by a a-
secretase to generate cytoplasmic APPsa and transmembrane C83 fragments. Both
remaining transmembrane fragments, C99 and C83, can be further cleaved by a y-
secretase, leading to the release and secretion of Alzheimer's related A(3 and
a non-
pathogenic peptide, p3, respectively (Vassar et al., 1999, Science, 286, 735-
741). Early
onset familial Alzheimer's disease is characterized by mutant APP protein with
a Met to
Leu substitution at position P1, characterized as the "Swedish" familial
mutation (Mullan
et al., 1992, Nature Genet., 1, 345). This APP mutation is characterized by a
dramatic
enhancement in (3-secretase cleavage (Citron et al., 1992, Nature, 360, 672).
The identification of (3-secretase, and y-secretase constituents involved in
the release
of (3-amyloid protein is of primary importance in the development of treatment
strategies
for Alzheimer's disease. Characterization of a-secretase is also important in
this regard
since a-secretase cleavage may compete with (3-secretase cleavage resulting in
non-
pathogenic vs. pathogenic protein production. Involvement of the two
metalloproteases,
ADAM 10, and TACE has been demonstrated in a-cleavage of AAP (Buxbaum et al.,
1999, J. Biol. Chem., 273, 27765, and Lammich et al., 1999, Proc. Natl. Acad.
Sci. U.S.A.,
96, 3922). Studies of y-secretase activity have demonstrated presenilin
dependence (De
Stooper et al., 1998, Nature, 391, 387, and De Stooper et al., 1999, Nature,
398, S 18), and
as such, presenilins have been proposed as y-secretase even though presenilin
does not
present proteolytic activity (Wolfe et al., 1999, Nature, 398, 513).
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Recently, Vassar et al., 1999, supra reported (3-secretase cleavage of AAP by
the
transmembrane aspartic protease beta site APP cleaving enzyme, BACE. While
other
potential candidates for ~i-secretase have been proposed (for review see Evin
et al., 1999,
Proc. Natl. Acad. Sci. U.S.A., 96, 3922), none have demonstrated the full
range of
5 characteristics expected from this enzyme. Vassar et al, supra, demonstrate
that BACE
expression and localization are as expected for ~3-secretase, that BACE
overexpression in
cells results in increased ~i-secretase cleavage of APP and Swedish APP, that
isolated
BACE demonstrates site specific proteolytic activity on APP derived peptide
substrates,
and that antisense mediated endogenous BACE inhibition results in dramatically
reduced
10 ~3-secretase activity.
Current treatment strategies for Alzheimer's disease rely on either the
prevention or
the alleviation of symptoms and/or the slowing down of disease progression.
Two drugs
approved in the treatment of Alzheimer's, donepezil (Aricept~) and tacrine
(Cognex~),
both cholinomimetics, attempt to slow the loss of cognitive ability by
increasing the
15 amount of acetylcholine available to the brain. Antioxidant therapy through
the use of
antioxidant compounds such as alpha-tocopherol (vitamin E), melatonin, and
selegeline
(Eldepryl~) attempt to slow disease progression by minimizing free radical
damage.
Estrogen replacement therapy is thought to incur a possible preventative
benefit in the
development of Alzheimer's disease based on limited data. The use of anti-
inflammatory
20 drugs may be associated with a reduced risk of Alzheimer's as well. Calcium
channel
blockers such as Nimodipine~ are considered to have a potential benefit in
treating
Alzheimer's disease due to protection of nerve cells from calcium overload,
thereby
prolonging nerve cell survival. Nootropic compounds, such as acetyl-L-
carnitine (Alcar~)
and insulin, have been proposed to have some benefit in treating Alzheimer's
due to
25 enhancement of cognitive and memory function based on cellular metabolism.
Whereby the above treatment strategies may all improve quality of life in
Alzheimer's patients, there exists an unmet need in the comprehensive
treatment and
prevention of this disease. As such, there exists the need for therapeutics
effective in
reversing the physiological changes associated with Alzheimer's disease,
specifically,
30 therapeutics that can eliminate and/or reverse the deposition of amyloid (3
peptide. The
use of compounds to modulate the expression of proteases that are instrumental
in the
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release of amyloid ~3 peptide, namely (3-secretase (BACE), and y-secretase
(presenilin), is
of therapeutic significance.
Tsai et al., 1999, Book of Abstrasts, 218th ACS National Meeting, New Orleans,
Aug 22-26, describe substrate-based alpha-aminoisobutyric acid derivatives of
difluoro
ketone peptidomimetic inhibitors of amyloid ~i peptide through y-secretase
inhibition.
Czech et al., International PCT publication No. W0/9921886, describe peptides
capable of inhibiting the interaction between presenilins and the (3-amyloid
peptide or its
precursor for therapeutic use.
Fournier et al., International PCT publication No. W0/9916874, describe human
brain proteins capable of interacting with presenilins and cDNAs encoding them
toward
therapeutic use.
St. George-Hyslop et al., International PCT publication No. W0/9727296,
describe
genes for proteins that interact with presenilins and their role in
Alzheimer's disease
toward therapeutic use.
Vassar et al., 1999, Science, 286, 735-741, describe specific antisense
oligonucleotides targeting BACE, used for inhibition studies of endogenous
BACE
expression in 101 cells and APPsw cells via lipid mediated transfection.
Vassar et al., 1999, Science, 286, 735-741, describe a cell culture model for
studying
BACE inhibition. Specific antisense nucleic acid molecules targeting BACE mRNA
were
used for inhibition studies of endogenous BACE expression in 101 cells and
APPsw
(Swedish type amyloid precursor protein expressing) cells via lipid mediated
transfection.
Antisense treatment resulted in dramatic reduction of both BACE mRNA by
Northern blot
analysis, and APPs~3sw ("Swedish" type (3-secretase cleavage product) by
ELISA, with
maximum inhibition of both parameters at 75-80%. This model was also used to
study the
effect of BACE inhibition on amyloid (3-peptide production in APPsw cells.
Games et al., 1995, Nature, 373, 523-527, describe a transgenic mouse model in
which mutant human familial type APP (Phe 717 instead of Val) is
overexpressed. This
model results in mice that progressively develop many of the pathological
hallmarks of
Alzheimer's disease, and as such, provides a model for testing therapeutic
drugs.
Particular degenerative and disease states that can be associated with BACE
expression modulation include but are not limited to Alzheimer's disease and
dementia.
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Donepezil, tacrine, selegeline, and acetyl-L-carnitine are non-limiting
examples of
pharmaceutical agents that can be combined with or used in conjunction with
the nucleic
acid molecules (e.g. ribozymes and antisense molecules) of the instant
invention. Those
skilled in the art will recognize that other drugs such as diuretic and
antihypertensive
compounds and therapies can be similarly be readily combined with the nucleic
acid
molecules of the instant invention (e.g. ribozymes and antisense molecules)
are hence
within the scope of the instant invention.
Identification of Potential Target Sites in Human BACE RNA
The sequence of human BACE was screened for accessible sites using a computer-
folding algorithm. Regions of the RNA that did not form secondary folding
structures and
contained potential ribozyme and/or antisense binding/cleavage sites were
identified. The
sequences of these cleavage sites are shown in Tables 18-23.
Selection of Enzymatic Nucleic Acid Cleavage Sites in Human BACE RNA
Ribozyme target sites were chosen by analyzing sequences of Human BACE
(Genbank sequence accession number: AF190725) and prioritizing the sites on
the basis
of folding. Ribozymes were designed that could bind each target and were
individually
analyzed by computer folding (Christoffersen et al., 1994 J. Mol. Struc.
Theochem, 311,
273; Jaeger et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess
whether the
ribozyme sequences fold into the appropriate secondary structure. Those
ribozymes with
unfavorable intramolecular interactions between the binding arms and the
catalytic core
were eliminated from consideration. As noted below, varying binding arm
lengths can be
chosen to optimize activity. Generally, at least 5 bases on each arm are able
to bind to, or
otherwise interact with, the target RNA.
Chemical Synthesis and Purification of Riboz~~nes and Antisense for Efficient
Cleavage
and/or blocking_of BACE RNA
Ribozymes and antisense constructs were designed to anneal to various sites in
the
RNA message. The binding arms of the ribozymes are complementary to the target
site
sequences described above, while the antisense constructs are fully
complimentary to the
target site sequences described above. The ribozymes and antisense constructs
were
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73
chemically synthesized. The method of synthesis used followed the procedure
for normal
RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. Soc.,
109,
7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et
al., supra, and
made use of common nucleic acid protecting and coupling groups, such as
dimethoxytrityl
at the 5'-end, and phosphoramidites at the 3'-end. The average stepwise
coupling yields
were >98%.
Ribozymes and antisense constructs were also synthesized from DNA templates
using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods
Enzymol. 180, 51). Ribozymes and antisense constructs were purified by gel
electrophoresis using general methods or were purified by high pressure liquid
chromatography (HPLC; See Wincott et al., supra; the totality of which is
hereby
incorporated herein by reference) and were resuspended in water. The sequences
of the
chemically synthesized ribozymes and antisense constructs used in this study
are shown
below in Table 18-23.
RibozXme Cleavage of BACE RNA Target in vitro
Ribozymes targeted to the human BACE RNA are designed and synthesized as
described above. These ribozymes can be tested for cleavage activity in vitro,
for
example, using the following procedure. The target sequences and the
nucleotide location
within the BACE RNA are given in Tables 18-23.
Cleavage Reactions: Full-length or partially full-length, internally-labeled
target
RNA for ribozyme cleavage assay is prepared by in vitro transcription in the
presence of
[a-32p] CTP, passed over a G SO Sephadex column by spin chromatography and
used as
substrate RNA without further purification. Alternately, substrates are 5'-32P-
end labeled
using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a
2X
concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-
HCI, pH 7.5
at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by adding
the 2X
ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was
also
pre-warmed in cleavage buffer. As an initial screen, assays are carried out
for 1 hour at
37°C using a final concentration of either 40 nM or 1 mM ribozyme,
i.e., ribozyme excess.
The reaction is quenched by the addition of an equal volume of 95% formamide,
20 mM
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74
EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is
heated to 95~C for 2 minutes, quick chilled and loaded onto a denaturing
polyacrylamide
gel. Substrate RNA and the specific RNA cleavage products generated by
ribozyme
cleavage are visualized on an autoradiograph of the gel. The percentage of
cleavage is
determined by Phosphor Imager~ quantitation of bands representing the intact
substrate
and the cleavage products.
Example 5: Phospholamban
Cardiac disease leading to heart failure is the leading cause of combined
morbidity
and mortality in the developed world. Nearly twenty million people worldwide
suffer
from heart failure related disease. An estimated five million Americans are
afflicted with
congestive heart failure (CHF), with 400,000 new cases diagnosed each year. In
the US,
cardiac disease associated failure results in approximately 40,000 deaths per
year, and is
associated with an additional 250,000 deaths (Harnish, 1999, Drug & Market
Development, 10, 114-119). Heart failure related disease represents a major
public health
issue due to an overall increase in prevalence and incidence in aging
populations with a
greater proportion of survivors of acute myocardial infarction (AMI) (Kannel
et al., 1994,
Br. Heart. J., 72 (supply, 3). Heart failure related disease represents the
most common
reason for hospitalization of elderly patients in the US. The resulting life
expectancy of
these patients is less than that of many common cancers, with five year
survival rates for
men and women at only 25% and 38% respectively, and with one year mortality
rates for
severe heart failure at 50% (Ho et al., 1993, Circulation, 88, 107).
Heart disease is characterized by a progressive decrease in cardiac output
resulting
from insufficient pumping activity of the diseased heart. The resulting venous
back-
pressure results in peripheral and pulmonary dysfunctional congestion. The
heart responds
to a variety of mechanical, hemodynamic, hormonal, and pathological stimuli by
increasing muscle mass in response to an increased demand for cardiac output.
The
resulting transformation of heart tissue (myocardial hypertrophy) can arise as
a result of
genetic, physiologic, and environmental factors, and represents an early
indication of
clinical heart disease and an important risk factor for subsequent heart
failure (Hunter and
Chien, 1999, New England J. of Medicine, 99, 313-322).
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Coronary heart disease is a predominant factor in the development of the
cardiac
disease state, along with prior AMI, hypertension, diabetes mellitus, and
valvular heart
disease. Diagnosis of cardiac disease includes determination of coronary heart
disease
associated left ventricular systolic dysfunction (LVSD) and/or left
ventricular diastolic
5 dysfunction (LVDD) by echocaardiographic imaging (Cleland, 1997, Dis
Management
Health Outcomes, 1, 169). Promising diagnosis may also rely on assaying atrial
natriuretic
peptide (ANP) and brain natriuretic peptide (BNP) concentrations. ANP and BNP
levels
are indicative of the level of ventricular dysfunction (Davidson et al., 1996,
Am. J.
Cardiol., 77, 828).
10 Current treatment strategies for cardiac disease associated failure are
varied.
Diuretics are often used to reduce pulmonary edema and dyspnea in patients
with fluid
overload, and are usually used in conjunction with angiotensin converting
enzyme (ACE)
inhibitors for vasodilation. Digoxin is another popular choice for treating
cardiac disease
as an ionotropic agent, however, doubts remain concerning the long-term
efficacy and
15 safety of Digoxin (Harnish, 1999, Drug & Market Development, 10, 114-119).
Carvedilol,
a beta-blocker, has been introduced to complement the above treatments in
order to slow
down the progression of cardiac disease. Antiarrhythmic agents can be used in
order to
reduce the risk of sudden death in patients suffering from cardiac disease.
Lastly, heart
transplants have been effective in the treatment of patients with advanced
stages of cardiac
20 disease, however, the limited supply of donor hearts greatly limits the
scope of this
treatment to the broad population (Harnish, 1999, Drug & Market Development,
10, 114-
119).
Whereby the above treatment strategies can all improve morbidity and mortality
associated with cardiac disease, the only existing definitive approach to
curing the diseased
25 heart is replacement by transplant. Even a healthy, transplanted heart can
become diseased
in response to the various stresses of mechanical, hemodynamic, hormonal, and
pathological stimuli associated with extrinsic risk factors. As such there
exists the need
for therapeutics effective in reversing the physiological changes associated
with cardiac
disease.
30 Myocardial hypertrophy and apoptosis are the underlying degenerative
process
associated with cardiac hypertrophy and failure. A variety of signaling
pathways are
involved in the progression of myocardial hypertrophy and myocardial
apoptosis. Genetic
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76
studies have been instrumental in elucidating these pathways and their
involvement in
cardiac disease through in vitro assays of cardiac muscle cells and in vivo
studies of
genetically engineered animals: . .
Studies in which the expression of specific genes have been altered in cardiac
myocytes have shown that specific peptide hormones, growth factors, and
cytokines can
activate various features of the hypertrophic response (Hunter and Chien,
1999, New
England J of Medicine, 99, 313-322). Particular substances that have been
characterized
from these studies include potential therapeutic and molecular targets
involved in heart
failure. Hunter et al., in Chien, KR, ed. Molecular basis of heart disease: a
companion
to Braunwald's Heart Disease, Philadelphia: W.B. Saunders, 1999:211-250,
describe
classes of therapeutic and molecular targets involved in heart failure
including:
1. Endothelin 1 and angiotensin II receptor antagonists, and antagonists of
ras, p38, and
c-jun N-terminal kinase (JNK) for inhibition of pathologic hypertrophy.
2. Insulin like growth factor I and growth hormone receptor stimulation for
promotion of
physiologic hypertrophy.
3. beta-1-adrenergic receptor blockers for inhibition of neurohumoral over
stimulation.
4. Phospholamban and Sarcolipin small molecule inhibitors for relief of
sarcoplasmic
reticulum calcium ATPase inhibition to provide enhancement of myocardial
contractile
and relaxation responses.
5. Small molecule inhibitors of (3-adrenergic receptor kinase to counteract
the
desensitization of G protein coupled receptor kinases in order to provide
enhancement
of myocardial contractile and relaxation responses.
6. Enhancement of angiogenic growth factors (VEGF, FGF-5) for relief of energy
deprivation in cardiac tissues.
7. Promoters of myocyte survival including gp 130 ligands (cardiotrophin 1),
and
Neuregulin for the inhibition of apoptosis of myocytes.
8. Inhibitors of apoptosis such as Caspase inhibitors for the inhibition of
apoptosis of
myocytes.
9. Inhibitors of cytokines such as TNF-alpha for the inhibition of apoptosis
of myocytes.
Congestive heart failure, heart failure, dilated cardiomyopathy and pressure
overload
hypertrophy are nonlimiting examples of disorders and disease states that can
be
associated with the above classes of molecular targets.
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The failure of cardiac contractile performance leading to cardiac disorders
and
disease, governed by impairment of cardiac excitation/contraction coupling,
points to the
importance of the signaling pathways involved in this process. The release and
uptake of
cytosolic Ca2+ by the sarcoplasmic reticulum plays an integral role in each
cycle of cardiac
contraction and excitation (Minamisawa et al., 1999, Cell, 99, 313-322). The
process of
Ca2+ reuptake is mediated by the cardiac sarcoplasmic reticulum Ca2+ ATPase
(SERCA2a). SERCA2a activity is regulated by phospholamban, a p52 muscle
specific
sarcoplasmic reticulum phosphoprotein (Koss et al., 1996, Circ. Res., 79, 1059-
1063, and
Simmerman et al., 1998, Physiol. Rev., 78, 921-947). In its active,
unphosphorylated state,
phospholamban is a potent inhibitor of SERCA2a activity. Phosphorylation of
phospholamban at serine 16 by cyclic AMP-dependent protein kinase (PKA) or
calmodulin kinase, results in the inhibition of phospholamban interaction with
SERCA2a.
This phosphorylation event is predominantly responsible for the proportional
increase in
the rate of Ca2+ uptake into the sarcoplasmic reticulum and resultant
ventricular relaxation
(Tada et al., 1982, Mol. Cell. Biochem., 46, 73-95, and Luo et al., 1998, J.
Biol. Chem.,
273, 4734-4739).
Since a proportional decrease in Ca2+ uptake is a hallmark feature of heart
failure
(Sordahl et al., 1973, Am. J. Physiol., 224, 497-502) and since an increase in
the relative
ratio of phospholamban to SERCA2a is an important determinant of sarcoplasmic
reticulum dysfunction in heart failure (Hasenfuss, 1998, Cardiovasc. Res., 37,
279-289),
'the targeting of phospholamban and related regulatory factors as therapeutic
targets for
heart disorders should prove valuable for cardiac indications.
Pystynen et al., International PCT publication No. WO 99/00132, describe
bisethers
of 1-oxa, aza and thianaphthalen-2-ones as small molecule inhibitors of
phospholamban
for increasing coronary flow via direct dilation of the coronary arteries.
Pystynen et al., International PCT publication No. WO 99/15523, describe
bisethers
of 1-oxa, aza and thianaphthalen-2-ones as small molecule inhibitors of
phospholamban
that are useful for treating heart failure.
The efficacy of the above mentioned treatment strategies is limited. Small
molecule
inhibition of a molecular target is often limited by toxicity, which can
restrict dosing and
overall efficacy.
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He et al., 1999, Circulation, 100, 974-980, describe endogenous expression of
mutant phospholamban and phospholamban antisense RNA to investigate the
corresponding effect on SERCA2a activity and cardiac myocyte contractility.
A more attractive approach to the treatment of heart disease would involve the
use of
ribozymes and/or antisense constructs to modulate the expression of target
molecules
involved in heart failure. The use of nucleic acid molecules of the instant
invention
permits highly specific regulation of the molecular targets of interest,
including
phospholamban (PLN) (GenBank accession No.1VM-002667), sarcolipin (SLN)
(GenBank accession No. NM_003063), angiotensin II receptor (GenBank accession
No.
U20860), endothelin 1 receptor (GenBank accession No. NM_001957), K-ras
(GenBank
accession No. NM-004985), p38 (GenBank accession No. AF092535), c-jun N-
terminal
kinase (GenBank accession No. NM_002750, L31951, NM 002753), growth hormone
receptor (GenBank accession No. NM_000163), insulin-like growth factor I
receptor
(GenBank accession No. NM_000875), beta-1-adrenergic receptor (GenBank
accession
No. NM_000024), (31-adrenergic receptor kinase (GenBank accession No.
NM_001619,
NM 005160), VEGF receptor (GenBank accession No. U43368, M27281 X15997),
fibroblast growth factor 5 (GenBank accession No. NM_004464), cardiotrophin I
(GenBank accession No. NM_001330), neuregulin (GenBank accession No.
AF009227),
TNF-alpha (GenBank accession No. X02910 X02159), PI3 kinase (GenBank accession
No. NM_006218, NM_006219, U86453, NM_002649, M61906), and AKT kinase
(GenBank accession No. NM_005163, M77198).
Various methods have been developed to assay phospholamban activity in vitro
and
in vivo. Holt et al., 1999, J. Mol. Cell. Cardiol., 31, 645-656, describe a
cell culture model
in which thyroid hormone control of contraction and the Ca2+-
ATPase/phospholamban
complex is studied in adult rat ventricular myocytes. Slack et al. 1997, J.
Biol. Chem.,
272, 18862-18868, describe studies in which the ectopic expression of
phospholamban in
mouse fast-twitch skeletal muscle cells alters sarcoplasmic reticulum Ca2+
transport and
muscle relaxation. MacLennan et al., 1996, Soc. Gen. Physiol. Ser., 51, 89-
103, in a
review of regulatory interactions between calcium ATPases and phospholamban
describe
phospholamban/ Ca2+-ATPase interactions in protein expressed in heterologous
cell
culture experiments. Cornwell et al., 1991, Mol. Pharmacol., 40,923-931,
describe the
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79
regulation of sarcoplasmic reticulum protein phosphorylation by localized
cyclic GMP-
dependent protein kinase in vascular smooth muscle cells.
Minamisawa et al., 1999, Cell, 99, 313-322, describe a phospholamban knockout
mouse model which affords protection from induced dilated cardiomyopathy.
Dillmann et
al., 1999, Am. J. Cardiol., 83, 89H-91H, describe a transgenic rat model for
the study of
altered expression of calcium regulatory proteins, including phospholamban,
and their
effect on myocyte contractile response. LekanneDeprez et al., 1998, J. Mol.
Cell.
Cardiol., 30, 1877-1888, describe a rat pressure-overload model to investigate
alterations
in gene expression of phospholamban, atrial natriuretic peptide (ANP),
sarcoplasmic
endoplasmic reticular calcium ATPase 2 (SERCA2), collagen Illal, and
calsequestrin
(CSQ). Jones et al., 1998, J. Clin. Invest., 101, 1385-1393, describe a mouse
model for
investigating the regulation of calcium signaling in transgenic mouse cardiac
myocytes
overexpressing calsequestrin. In this study, the upregulation and
downregulation of
calcium uptake and release proteins were determined, including phospholamban.
Lorenz
et al., 1997, Am .l. Physiol., 273, 6, describe a mouse model for the study of
regulatory
effects of phospholamban on cardiac function in intact mice. This study makes
use of
animal models with altered levels of phospholamban to permit in vivo
evaluation of the
physiological role of phospholamban. Arai et al., 1996, Saishin Igaku, S 1,
1095-1104,
presents a review article of gene targeted animal models expressing
cardiovascular
abnormalities. The study of phospholamban and other protein expression
modification
effects in mice is presented. Wankerl et al., 1995, J. Mol. Med., 73, 487-496,
presents a
review article describing the study of calcium transport proteins in the
nonfailing and
failing heart. Animal models investigating the major calcium handling
myocardial
proteins, including phospholamban, are described. These models, as well as
others, may
be used to evaluate the effect of treatment with nucleic acid molecules of the
instant
invention on cardiac function. Endpoints may be, but are not limited to, left
ventricular
pressure, left ventricular pressure as a function of time (LVdP/dt), and mean
arterial blood
pressure. Endpoints will be evaluated under basal and stimulated (cardiac
load)
conditions.
Particular degenerative and disease states that can be associated with
phospholamban expression modulation include but are not limited to congestive
heart
failure, heart failure, dilated cardiomyopathy and pressure overload
hypertrophy:
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Digoxin, Bendrofluazide, Dofetilide, and Carvedilol are non-limiting examples
of
pharmaceutical agents that can be combined with or used in conjunction with
the nucleic
acid molecules (e.g. ribozymes and antisense molecules) of the instant
invention. Those
skilled in the art will recognize that other drugs such as diuretic and
antihypertensive
5 compounds and therapies can be similarly be readily combined with the
nucleic acid
molecules of the instant invention (e.g. ribozymes and antisense molecules)
are hence
within the scope of the instant invention.
Identification of Potential Target Sites in Human phospholamban RNA
10 The sequence of human phospholamban was screened for accessible sites using
a
computer folding algorithm. Regions of the RNA that did not form secondary
folding
structures and contained potential ribozyme and/or antisense binding/cleavage
sites were
identified. The sequences of these cleavage sites are shown in Tables 24-30.
15 Selection of Enzymatic Nucleic Acid Cleavage Sites in Human phospholamban
RNA
Ribozyme target sites were chosen by analyzing sequences of Human
phospholamban (Genbank sequence accession number: NM_002667) and prioritizing
the
sites on the basis of folding. Ribozymes were designed that could bind each
target and
were individually analyzed by computer folding (Christoffersen et al., 1994 J.
Mol. Struc.
20 Theochem, 311, 273; Jaeger et al., 1989, Proc. Natl. Acad. Sci. USA, 86,
7706) to assess
whether the ribozyme sequences fold into the appropriate secondary structure.
Those
ribozymes with unfavorable intramolecular interactions between the binding
arms and the
catalytic core were eliminated from consideration. As noted below, varying
binding arm
lengths can be chosen to optimize activity. Generally, at least 5 bases on
each arm are able
25 to bind to, or otherwise interact with, the target RNA.
Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient
Cleavage
and/or blocking of phospholamban RNA
Ribozymes and antisense constructs were designed to anneal to various sites in
the
30 RNA message. The binding arms of the ribozymes are complementary to the
target site
sequences described above, while the antisense constructs are fully
complimentary to the
target site sequences described above. The ribozymes and antisense constructs
were
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chemically synthesized. The method of synthesis used followed the procedure
for normal
RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. Soc.,
109,
7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et
al., supra, and
made use of common nucleic acid protecting and coupling groups, such as
dimethoxytrityl
at the 5'-end, and phosphoramidites at the 3'-end. The average stepwise
coupling yields
were >98%.
Ribozymes and antisense constructs were also synthesized from DNA templates
using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods
Enzymol. 180, 51). Ribozymes and antisense constructs were purified by gel
0 electrophoresis using general methods or were purified by high pressure
liquid
chromatography (HPLC; see Wincott et al., supra; the totality of which is
hereby
incorporated herein by reference) and were resuspended in water. The sequences
of the
chemically synthesized ribozymes and antisense constructs used in this study
are shown
below in Table 24-30.
5
Ribozyme Cleavage of phospholamban RNA Target in vitro
Ribozymes targeted to the human phospholamban RNA are designed and
synthesized as described above. These ribozymes can be tested for cleavage
activity in
vitro, for example using the following procedure. The target sequences and the
nucleotide
0 location within the phospholamban RNA are given in Tables 24-30.
Cleavage Reactions: Full-length or partially full-length, internally-labeled
target
RNA for ribozyme cleavage assay is prepared by in vitro transcription in the
presence of
[a-32p] CTP, passed over a G 50 Sephadex column by spin chromatography and
used as
substrate RNA without further purification. Alternately, substrates are 5'-32P-
end labeled
5 using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a
2X
concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-
HCI, pH 7.5
at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by adding
the 2X
ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was
also
pre-warmed in cleavage buffer. As an initial screen, assays are carned out for
1 hour at
0 37°C using a final concentration of either 40 nM or 1 mM ribozyme,
i.e., ribozyme excess.
The reaction is quenched by the addition of an equal volume of 95% formamide,
20 mM
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EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is
heated to 95~C for 2 minutes, quick chilled and loaded onto a denaturing
polyacrylamide
gel. Substrate RNA and the specific RNA cleavage products generated by
ribozyme
cleavage are visualized on an autoradiograph of the gel. The percentage of
cleavage is
determined by Phosphor Imager~ quantitation of bands representing the intact
substrate
and the cleavage products.
Tissue distribution of BrdU-labeled antisense in mice
CD1 mice were injected with a single bolus (30 mg/kg) of a BrdU-labeled
antisense
oligonucleotide or a similar molar amount of BrdU (as a control). At various
time points
(30 min, 2h and 6 h), mice were sacrificed and major tissues isolated and
fixed.
Distribution of antisense oligonucleotides was determined by probing with an
anti-BrdU
antibody and immunohistochemical staining. Tissue slices were probed with an
anti-BrdU
antibody followed by a reporter enzyme-conjugated second antibody and finally
an enzyme
substrate. Visualization of the colored product by microscopy indicated
nuclear staining,
demonstrating effective distribution of antisense oligonucleotide in cardiac
tissue.
Tissue distribution of BrdU-labeled ribozymes in monkey
Rhesus monkeys were dosed with BrdU-labeled ribozyme by intravenous bolus
~0 injection at 0.1, 1.0, and 10 mg/kg once daily over five days. Saline
injection was used in
control animals. Animals were sacrificed and major tissues isolated and fixed.
Tissue
samples were probed with an anti-BrdU antibody followed by a reporter enzyme-
conjugated second antibody and finally an enzyme substrate. Significant
quantities of
chemically modified ribozyme are detected in cardiac tissue following this
dosing regimen.
?5
Example 6: HBV
Chronic hepatitis B is caused by an enveloped virus, commonly known as the
hepatitis B virus or HBV. HBV is transmitted via infected blood or other body
fluids,
especially saliva and semen, during delivery, sexual activity, or sharing of
needles
30 contaminated by infected blood. Individuals may be "carners" and transmit
the infection
to others without ever having experienced symptoms of the disease. Persons at
highest
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risk are those with multiple sex partners, those with a history of sexually
transmitted
diseases, parenteral drug users, infants born to infected mothers, "close"
contacts or sexual
partners of infected persons, and healthcare personnel or other service
employees who
have contact with blood. Transmission is also possible via tattooing, ear or
body piercing,
and acupuncture; the virus is also stable on razors, toothbrushes, baby
bottles, eating
utensils, and some hospital equipment such as respirators, scopes and
instruments. There
is no evidence that HBsAg positive food handlers pose a health risk in an
occupational
setting, nor should they be excluded from work. Hepatitis B has never been
documented
as being a food-borne disease. The average incubation period is 60 to 90 days,
with a
I 0 range of 45 to 180; the number of days appears to be related to the amount
of virus to
which the person was exposed. However, determining the length of incubation is
difficult,
since onset of symptoms is insidious. Approximately 50% of patients develop
symptoms
of acute hepatitis that last from 1 to 4 weeks. Two percent or less of these
individuals
develop fulminant hepatitis resulting in liver failure and death.
5 The determinants of severity include: (1) The size of the dose to which the
person
was exposed; (2) the person's age with younger patients experiencing a milder
form of the
disease; (3) the status of the immune system with those who are
immunosuppressed
experiencing milder cases; and (4) the presence or absence of co-infection
with the Delta
virus (hepatitis D), with more severe cases resulting from co-infection. In
symptomatic
!0 cases, clinical signs include loss of appetite, nausea, vomiting, abdominal
pain in the right
upper quadrant, arthralgia, and tiredness/loss of energy. Jaundice is not
experienced in all
cases, however, jaundice is more likely to occur if the infection is due to
transfusion or
percutaneous serum transfer, and it is accompanied by mild pruritus in some
patients.
Bilirubin elevations are demonstrated in dark urine and clay-colored stools,
and liver
!5 enlargement may occur accompanied by right upper-quadrant pain. The acute
phase of the
disease may be accompanied by severe depression, meningitis, Guillain-Barre
syndrome,
myelitis, encephalitis, agranulocytosis, and/or thrombocytopenia.
Hepatitis B is generally self limiting and will resolve in approximately 6
months.
Asymptomatic cases can be detected by serologic testing, since the presence of
the virus
.0 leads to production of large amounts of HBsAg in the blood. This antigen is
the first and
most useful diagnostic marker for active infections. However, if HBsAg remains
positive
for 20 weeks or longer, the person is likely to remain positive indefinitely
and is now a
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carrier. While only 10% of persons over age 6 who contract HBV become
carriers, 90% of
infants infected during the first year of life do so.
Hepatitis B virus (HBV) infects over 300 million people worldwide (Imperial,
1999,
Gastroenterol. Hepatol., 14 (supply, S1-5). In the United States approximately
1.25
million individuals are chronic carriers of HBV as evidenced by the fact that
they have
measurable hepatitis B virus surface antigen HBsAg in their blood. The risk of
becoming
a chronic HBsAg carrier is dependent upon the mode of acquisition of infection
as well as
the age of the individual at the time of infection. For those individuals with
high levels of
viral replication, chronic active hepatitis with progression to cirrhosis,
liver failure and
0 hepatocellular carcinoma (HCC) is common, and liver transplantation is the
only treatment
option for patients with end-stage liver disease from HBV.
The natural progression of chronic HBV infection over a 10 to 20 year period
leads
to cirrhosis in 20-to-50% of patients and progression of HBV infection to
hepatocellular
carcinoma has been well documented. There have been no studies that have
determined
5 sub-populations that are most likely to progress to cirrhosis and/or
hepatocellular
carcinoma, thus all patients have equal risk of progression.
It is important to note that the survival for patients diagnosed with
hepatocellular
carcinoma is only 0.9 to 12.8 months from initial diagnosis (Takahashi et al.,
1993,
American Journal of Gastroenterology, 88, 240-243). Treatment of
hepatocellular
.0 carcinoma with chemotherapeutic agents has not proven effective and only
10% of patients
will benefit from surgery due to extensive tumor invasion of the liver
(Trinchet et al.,
1994, Presse Medicine, 23, 831-833). Given the aggressive nature of primary
hepatocellular carcinoma, the only viable treatment alternative to surgery is
liver
transplantation (Pichlmayr et al., 1994, Hepatology., 20, 33S-40S).
5 Upon progression to cirrhosis, patients with chronic HCV infection present
with
clinical features, which are common to clinical cirrhosis regardless of the
initial cause
(D'Amico et al., 1986, Digestive Diseases and Sciences, 31, 468-475). These
clinical
features may include: bleeding esophageal varices, ascites, jaundice, and
encephalopathy
(Zakim D, Boyer TD. Hepatology a textbook of liver disease, Second Edition
Volume 1.
0 1990 W.B. Saunders Company. Philadelphia). In the early stages of cirrhosis,
patients are
classified as compensated, meaning that although liver tissue damage has
occurred, the
patient's liver is still able to detoxify metabolites in the blood-stream. In
addition, most
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patients with compensated liver disease are asymptomatic and the minority with
symptoms
report only minor symptoms such as dyspepsia and weakness. In the later stages
of
cirrhosis, patients are classified as decompensated meaning that their ability
to detoxify
metabolites in the bloodstream is diminished and it is at this stage that the
clinical features
5 described above will present.
In 1986, D'Amico et al. described the clinical manifestations and survival
rates in
1155 patients with both alcoholic and viral associated cirrhosis (D'Amico
supra). Of the
1155 patients, 435 (37%) had compensated disease although 70% were
asymptomatic at
the beginning of the study. The remaining 720 patients (63%) had decompensated
liver
I 0 disease with 78% presenting with a history of ascites, 31% with jaundice,
17% had
bleeding and 16% had encephalopathy. Hepatocellular carcinoma was observed in
six
(0.5%) patients with compensated disease and in 30 (2.6%) patients with
decompensated
disease.
Over the course of six years, the patients with compensated cirrhosis
developed
I 5 clinical features of decompensated disease at a rate of 10% per year. In
most cases, ascites
was the first presentation of decompensation. In addition, hepatocellular
carcinoma
developed in 59 patients who initially presented with compensated disease by
the end of
the six-year study.
With respect to survival, the D'Amico study indicated that the five-year
survival rate
!0 for all patients on the study was only 40%. The six-year survival rate for
the patients who
initially had compensated cirrhosis was 54% while the six-year survival rate
for patients
who initially presented with decompensated disease was only 21 %. There were
no
significant differences in the survival rates between the patients who had
alcoholic
cirrhosis and the patients with viral related cirrhosis. The major causes of
death for the
!5 patients in the D'Amico study were liver failure in 49%; hepatocellular
carcinoma in 22%;
and, bleeding in 13% (D'Amico supra).
Hepatitis B virus is a double-stranded circular DNA virus. It is a member of
the
Hepadnaviridae family. The virus consists of a central core that contains a
core antigen
(HBcAg) surrounded by an envelope containing a surface protein/surface antigen
(HBsAg)
.0 and is 42 nm in diameter. It also contains an a antigen (HBeAg) which,
along with HBcAg
and HBsAg, is helpful in identifying this disease
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In HBV virions, the genome is found in an incomplete double-stranded form. HBV
uses a reverse transcriptase to transcribe a positive-sense full length RNA
version of its
genome back into DNA. This reverse transcriptase also contains DNA polymerise
activity
and thus begins replicating the newly synthesized minus-sense DNA strand.
However, it
appears that the core protein encapsidates the reverse-
transcriptase/polymerase before it
completes replication.
From the free-floating form, the virus must first attach itself specifically
to a host
cell membrane. Viral attachment is one of the crucial steps which determines
host and
tissue specificity. However, currently there are no in vitro cell-lines that
can be infected by
HBV. There are some cells lines, such as HepG2, which can support viral
replication only
upon transient or stable transfection using HBV DNA.
After attachment, fusion of the viral envelope and host membrane must occur to
allow the viral core proteins containing the genome and polymerise to enter
the cell. Once
inside, the genome is translocated to the nucleus where it is repaired and
cyclized.
5 The complete closed circular DNA genome of HBV remains in the nucleus and
gives rise to four transcripts. These transcripts initiate at unique sites but
share the same
3'-ends. The 3.5-kb pregenomic RNA serves as a template for reverse
transcription and
also encodes the nucleocapsid protein and polymerise. A subclass of this
transcript with a
5'-end extension codes for the precore protein that, after processing, is
secreted as HBV a
!0 antigen. The 2.4-kb RNA encompasses the pre-S 1 open reading frame (ORF)
that encodes
the large surface protein. The 2.1-kb RNA encompasses the pre-S2 and S ORFs
that
encode the middle and small surface proteins, respectively. The smallest
transcript (~0.8-
kb) codes for the X protein, a transcriptional activator.
Multiplication of the HBV genome begins within the nucleus of an infected
cell.
!5 RNA polymerise II transcribes the circular HBV DNA into greater-than-full
length
mRNA. Since the mRNA is longer than the actual complete circular DNA,
redundant
ends are formed. Once produced, the pregenomic RNA exits the nucleus and
enters the
cytoplasm.
The packaging of pregenomic RNA into core particles is triggered by the
binding of
.0 the HBV polymerise to the 5' epsilon stem-loop. RNA encapsidation is
believed to occur
as soon as binding occurs. The HBV polymerise also appears to require
associated core
protein in order to function. The HBV polymerise initiates reverse
transcription from the
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5' epsilon stem-loop three to four base pairs at which point the polymerise
and attached
nascent DNA are transferred to the 3' copy of the DR1 region. Once there, the
(-)DNA is
extended by the HBV polymerise while the RNA template is degraded by the HBV
polymerise RNAse H activity. When the HBV polymerise reaches the 5' end, a
small
stretch of RNA is left undigested by the RNAse H activity. This segment of RNA
is
comprised of a small sequence just upstream and including the DR1 region. The
RNA
oligomer is then translocated and annealed to the DR2 region at the 5' end of
the (-)DNA.
It is used as a primer for the (+)DNA synthesis which is also generated by the
HBV
polymerise. It appears that the reverse transcription as well as plus strand
synthesis may
I 0 occur in the completed core particle.
Since the pregenomic RNA is required as a template for DNA synthesis, this RNA
is
an excellent target for ribozyme cleavage. Nucleoside analogues that have been
documented to inhibit HBV replication target the reverse transcriptase
activity needed to
convert the pregenomic RNA into DNA. Ribozyme cleavage of the pregenomic RNA
I 5 template would be expected to result in a similar inhibition of HBV
replication. Further,
targeting the 3'-end of the pregenomic RNA that is common to all HBV
transcripts could
result in reduction of all HBV gene products and an additional level of
inhibition of HBV
replication.
As previously mentioned, HBV does not infect cells in culture. However,
!0 transfection of HBV DNA (either as a head-to-tail dimer or as an
"overlength" genome of
>100%) into HuH7 or Hep G2 hepatocytes results in viral gene expression and
production
of HBV virions released into the media. Thus, HBV replication competent DNA
would be
co-transfected with ribozymes in cell culture. Such an approach has been used
to report
intracellular ribozyme activity against HBV (zu Putlitz, et al., 1999, J.
Yirol., 73, 5381-
!5 5387, and Kim et al., 1999, Biochem. Biophys. Res. Commun., 257, 759-765).
In addition,
stable hepatocyte cell lines have been generated that express HBV. In these
cells only
ribozyme would need to be delivered; however, a delivery screen would need to
be
performed. In addition, stable hepatocyte cell lines have been generated that
express HBV.
Intracellular HBV gene expression can be assayed by a Taqman~ assay for HBV
.0 RNA or by ELISA for HBV protein. Extracellular virus can be assayed by PCR
for DNA
or ELISA for protein. Antibodies are commercially available for HBV surface
antigen and
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core protein. A secreted alkaline phosphatase expression plasmid can be used
to
normalize for differences in transfection efficiency and sample recovery.
There are several small animal models to study HBV replication. One is the
transplantation of HBV-infected liver tissue into irradiated mice. Viremia (as
evidenced
by measuring HBV DNA by PCR) is first detected 8 days after transplantation
and peaks
between 18 - 25 days (Ilan et al., 1999, Hepatology, 29, 553-562).
Transgenic mice that express HBV have also been used as a model to evaluate
potential anti-virals. HBV DNA is detectable in both liver and serum (Morrey
et al., 1999,
Antiviral Res., 42, 97-108).
I 0 An additional model is to establish subcutaneous tumors in nude mice with
Hep G2
cells transfected with HBV. Tumors develop in about 2 weeks after inoculation
and
express HBV surface and core antigens. HBV DNA and surface antigen is also
detected in
the circulation of tumor-bearing mice (Yao et al., 1996, J. Viral Hepat., 3,
19-22).
Woodchuck hepatitis virus (WHV) is closely related to HBV in its virus
structure,
I 5 genetic organization, and mechanism of replication. As with HBV in humans,
persistent
WHV infection is common in natural woodchuck populations and is associated
with
chronic hepatitis and hepatocellular carcinoma (HCC). Experimental studies
have
established that WHV causes HCC in woodchucks and woodchucks chronically
infected
with WHV have been used as a model to test a number of anti-viral agents. For
example,
!0 the nucleoside analogue 3T3 was observed to cause dose dependent reduction
in virus
(5O% reduction after two daily treatments at the highest dose) (Hurwitz et
al., 1998.
Antimicrob. Agents Chemother., 42, 2804-2809).
Current therapeutic goals of treatment are three-fold: to eliminate
infectivity and
transmission of HBV to others, to arrest the progression of liver disease and
improve the
!5 clinical prognosis, and to prevent the development of hepatocellular
carcinoma (HCC).
Interferon alpha use is the most common therapy for HBV; however, recently
Lamivudine (3TC) has been approved by the FDA. Interferon alpha (IFN-alpha) is
one
treatment for chronic hepatitis B. The standard duration of IFN-alpha therapy
is 16 weeks,
however, the optimal treatment length is still poorly defined. A complete
response (HBV
DNA negative HBeAg negative) occurs in approximately 25% of patients. Several
factors
have been identified that predict a favorable response to therapy including:
High ALT ,
low HBV DNA , being female, and heterosexual orientation.
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There is also a risk of reactivation of the hepatitis B virus even after a
successful
response, this occurs in around 5% of responders and normally occurs within 1
year.
Side effects resulting from treatment with type 1 interferons can be divided
into four
general categories including: Influenza-like symptoms, neuropsychiatric,
laboratory
abnormalities, and other miscellaneous side effects. Examples of influenza-
like
symptoms include, fatigue, fever; myalgia, malaise, appetite loss,
tachycardia, rigors,
headache and arthralgias. The influenza-like symptoms are usually short-lived
and tend to
abate after the first four weeks of dosing (Dusheiko et al., 1994, .lournal of
Viral Hepatitis,
1, 3-5). Neuropsychiatric side effects include irritability, apathy, mood
changes, insomnia,
I 0 cognitive changes, and depression. Laboratory abnormalities include the
reduction of
myeloid cells, including granulocytes, platelets and to a lesser extent, red
blood cells.
These changes in blood cell counts rarely lead to any significant clinical
sequellae. In
addition, increases in triglyceride concentrations and elevations in serum
alaine and
aspartate aminotransferase concentration have been observed. Finally, thyroid
I 5 abnormalities have been reported. These thyroid abnormalities are usually
reversible after
cessation of interferon therapy and can be controlled with appropriate
medication while on
therapy. Miscellaneous side effects include nausea, diarrhea, abdominal and
back pain,
pruritus, alopecia, and rhinorrhea. In general, most side effects will abate
after 4 to 8
weeks of therapy (Dushieko et al., supra ).
!0 Lamivudine (3TC) is a nucleoside analogue, which is a very potent and
specific
inhibitor of HBV DNA synthesis. Lamivudine has recently been approved for the
treatment of chronic Hepatitis B. Unlike treatment with interferon, treatment
with 3TC
does not eliminate the HBV from the patient. Rather, viral replication is
controlled and
chronic administration results in improvements in liver histology in over 50%
of patients.
!5 Phase III studies with 3TC, showed that treatment for one year was
associated with
reduced liver inflammation and a delay in scarnng of the liver. In addition,
patients treated
with Lamivudine (100mg per day) had a 98 percent reduction in hepatitis B DNA
and a
significantly higher rate of seroconversion, suggesting disease improvements
after
completion of therapy. However, stopping of therapy resulted in a reactivation
of HBV
replication in most patients. In addition recent reports have documented 3TC
resistance in
approximately 30% of patients.
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Particular degenerative and disease states that can be associated with HBV
expression modulation include but are not limited to, HBV infection,
hepatitis, cancer,
tumorigenesis, cirrhosis, liver failure and others.
Lamivudine (3TC), L-FMAU, adefovir dipivoxil, type 1 Interferon, therapeutic
5 vaccines, steriods, and 2'-5' Oligoadenylates are non-limiting examples of
pharmaceutical
agents that can be combined with or used in conjunction with the nucleic acid
molecules
(e.g. ribozymes and antisense molecules) of the instant invention. Those
skilled in the art
will recognize that other drugs such as diuretic and antihypertensive
compounds or other
therapies can similarly and readily be combined with the nucleic acid
molecules of the
I 0 instant invention (e.g. ribozymes and antisense molecules) and are,
therefore, within the
scope of the instant invention.
Current therapies for treating HBV infection, including interferon and
nucleoside
analogues, are only partially effective. In addition, drug resistance to
nucleoside analogues
is now emerging, making treatment of chronic Hepatitis B more difficult. Thus,
a need
I 5 exists for effective treatment of this disease which utilizes antiviral
inhibitors which work
by mechanisms other than those currently utilized in the treatment of both
acute and
chronic hepatitis B infections.
Draper, US patent No. 6,017,756, describes the use of ribozymes for the
inhibition
of Hepatitis B Virus.
?0 Passman et al., 2000, Biochem. Biophys. Res. Commun., 268(3), 728-733.; Gan
et
al., 1998, J. Med. Coll. PLA, 13(3), 157-159.; Li et al., 1999, Jiefangjun
Yixue Zazhi,
24(2), 99-101.; Putlitz et al., 1999, J. Virol., 73(7), 5381-5387.; Kim et
al., 1999,
Biochem. Biophys. Res. Commun., 257(3), 759-765.; Xu et al., 1998, Bingdu
Xuebao,
14(4), 365-369.; Welch et al., 1997, Gene Ther., 4(7), 736-743.; Goldenberg et
al., 1997,
?5 International PCT publication No. WO 97/08309, Wands et al., 1997, J. of
Gastroenterology and Hepatology, 12(suppl.), 5354-5369.; Ruiz et al., 1997,
BioTechniques, 22(2), 338-345.; Gan et al., 1996, J. Med. Coll. PLA, 11(3),
171-175.;
Beck and Nassal, 1995, Nucleic Acids Res., 23(24), 4954-62.; Goldenberg, 1995,
International PCT publication No. WO 95/22600.; Xu et al., 1993, Bingdu
Xuebao, 9(4),
30 331-6.; Wang et al., 1993, Bingdu Xuebao, 9(3), 278-80, all describe
ribozymes that are
targeted to cleave a specific HBV target site.
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91
The enzymatic nucleic acid molecules of the instant invention exhibit a high
degree
of specificity for only the viral mRNA in infected cells. Nucleic acid
molecules of the
instant invention targeted to highly conserved sequence regions allow the
treatment of
many strains of human HBV with a single compound. No treatment presently
exists which
specifically attacks expression of the viral genes) that are responsible for
transformation
of hepatocytes by HBV.
The methods of this invention can be used to treat human hepatitis B virus
infections, which include productive virus infection, latent or persistent
virus infection,
and HBV-induced hepatocyte transformation. The utility can be extended to
other species
of HBV which infect non-human animals where such infections are of veterinary
importance.
Preferred target sites are genes required for viral replication, a non-
limiting example
includes genes for protein synthesis, such as the 5' most 1500 nucleotides of
the HBV
pregenomic mRNAs. For sequence references, see Renbao et al., 1987, Sci. Sin.,
30, 507.
This region controls the translational expression of the core protein (C), X
protein (X) and
DNA polymerase (P) genes and plays a role in the replication of the viral DNA
by serving
as a template for reverse transcriptase. Disruption of this region in the RNA
results in
deficient protein synthesis as well as incomplete DNA synthesis (and
inhibition of
transcription from the defective genomes). Target sequences 5' of the
encapsidation site
can result in the inclusion of the disrupted 3' RNA within the core virion
structure and
targeting sequences 3' of the encapsidation site can result in the reduction
in protein
expression from both the 3' and 5' fragments.
Alternative regions outside of the 5' most 1500 nucleotides of the pregenomic
mRNA also make suitable targets of enzymatic nucleic acid mediated inhibition
of HBV
replication. Such targets include the mRNA regions that encode the viral S
gene.
Selection of particular target regions will depend upon the secondary
structure of the
pregenomic mRNA. Targets in the minor mRNAs can also be used, especially when
folding or accessibility assays in these other RNAs reveal additional target
sequences that
are unavailable in the pregenomic mRNA species.
A desirable target in the pregenomic RNA is a proposed bipartite stem-loop
structure
in the 3'-end of the pregenomic RNA which is believed to be critical for viral
replication
(Kidd and Kidd-Ljunggren, 1996. Nuc. Acid Res. 24:3295-3302). The 5'end of the
HBV
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pregenomic RNA carnes a cis-acting encapsidation signal, which has inverted
repeat
sequences that are thought to form a bipartite stem-loop structure. Due to a
terminal
redundancy in the pregenomic RNA, the putative stem-loop also occurs at the 3'-
end.
While it is the 5' copy which functions in polymerase binding and
encapsidation, reverse
transcription actually begins from the 3' stem-loop. To start reverse
transcription, a 4 nt
primer which is covalently attached to the polymerase is made, using a bulge
in the 5'
encapsidation signal as template. This primer is then shifted, by an unknown
mechanism,
to the DR1 primer binding site in the 3' stem-loop structure, and reverse
transcription
proceeds from that point. The 3' stem-loop, and especially the DR1 primer
binding site,
I 0 appear to be highly effective targets for ribozyme intervention.
Sequences of the pregenomic RNA are shared by the mRNAs for surface, core,
polymerase, and X proteins. Due to the overlapping nature of the HBV
transcripts, all
share a common 3'-end. Ribozyme targeting this common 3'-end will thus cleave
the
pregenomic RNA as well as all of the mRNAs for surface, core, polymerase and X
5 proteins.
In preferred embodiments, the invention features a method for the analysis of
HBV
proteins. This method is useful in determining the efficacy of HBV inhibitors.
Specifically, the instant invention features an assay for the analysis of
HBsAg proteins and
secreted alkaline phosphatase (SEAP) control proteins to determine the
efficacy of agents
!0 used to modulate HBV expression.
The method consists of coating a micro-titer plate with an antibody such as
anti-
HBsAg Mab (for example, Biostride B88-95-3lad,ay) at 0.1 to 10 p,g/ml in a
buffer (for
example, carbonate buffer, such as NaZC03 15 mM, NaHC03 35 mM, pH 9.5) at
4°C
overnight. The microtiter wells are then washed with PBST or the equivalent
thereof, (for
'.5 example, PBS, 0.05% Tween 20) and blocked for 0.1-24 hr at 37° C
with PBST, 1% BSA
or the equivalent thereof. Following washing as above, the wells are dried
(for example, at
37° C for 30 min). Biotinylated goat anti-HBsAg or an equivalent
antibody (for example,
Accurate YVS1807) is diluted (for example at 1:1000) in PBST and incubated in
the wells
(for example, 1 hr. at 37° C). The wells are washed with PBST (for
example, 4x). A
~0 conjugate, (for example, Streptavidin/Alkaline Phosphatase Conjugate,
Pierce 21324) is
diluted to 10-10,000 ng/ml in PBST, and incubated in the wells (for example, 1
hr. at 37°
C). After washing as above, a substrate (for example, p-nitrophenyl phosphate
substrate,
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Pierce 37620) is added to the wells, which are then incubated (for example, 1
hr. at 37° C).
The optical density is then determined (for example, at 405 nm). SEAP levels
are then
assayed, for example, using the Great EscAPe~ Detection Kit (Clontech K2041-
1), as per
the manufacturers instructions. In the above example, incubation times and
reagent
concentrations may be varied to achieve optimum results, a non-limiting
example is
described in Example 6.
Comparison of this HBsAg ELISA method to a commercially available assay from
World Diagnostics, Inc. 15271 NW 60'h Ave, #201, Miami Lakes, FL 33014 (305)
827-
3304 (Cat. No. EL10018) demonstrates an increase in sensitivity (signal:noise)
of 3-20
fold.
Identification of Potential Target Sites in Human HBV RNA
The sequence of human HBV was screened for accessible sites using a computer-
folding algorithm. Regions of the RNA that did not form secondary folding
structures and
I 5 contained potential ribozyme and/or antisense binding/cleavage sites were
identified. The
sequences of these cleavage sites are shown in Tables 36-43.
Selection of Enzymatic Nucleic Acid Cleavage Sites in Human HBV RNA
Ribozyme target sites were chosen by analyzing sequences of Human HBV
!0 (accession number: AF100308.1) and prioritizing the sites on the basis of
folding.
Ribozymes were designed that could bind each target and were individually
analyzed by
computer folding (Christoffersen et al., 1994 J. Mol. Struc. Theochem, 31 l,
273; Jaeger et
al., 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the
ribozyme sequences
fold into the appropriate secondary structure. Those ribozymes with
unfavorable
!5 intramolecular interactions between the binding arms and the catalytic core
were
eliminated from consideration. As noted herein, varying binding arm lengths
can be
chosen to optimize activity. Generally, at least 5 bases on each arm are able
to bind to, or
otherwise interact with, the target RNA.
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Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient
Cleavage
and/or blocking of HBV RNA
Ribozymes and antisense constructs were designed to anneal to various sites in
the
RNA message. The binding arms of the ribozymes are complementary to the target
site
sequences described above, while the antisense constructs are fully
complementary to the
target site sequences described above. The ribozymes and antisense constructs
were
chemically synthesized. The method of synthesis used followed the procedure
for normal
RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. Soc.,
109,
7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et
al., supra, and
0 made use of common nucleic acid protecting and coupling groups, such as
dimethoxytrityl
at the 5'-end, and phosphoramidites at the 3'-end. The average stepwise
coupling yields
were typically >98%.
Ribozymes and antisense constructs were also synthesized from DNA templates
using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods
5 Enzymol. 180, 51). Ribozymes and antisense constructs were purified by gel
electrophoresis using general methods or were purified by high pressure liquid
chromatography (HPLC; see Wincott et al., supra; the totality of which is
hereby
incorporated herein by reference) and were resuspended in water. The sequences
of the
chemically synthesized ribozymes used in this study are shown below in Table
43.
'.0
Ribozyme Cleavage of HBV RNA Target in vitro
Ribozymes targeted to the human HBV RNA are designed and synthesized as
described above. These ribozymes can be tested for cleavage activity in vitro,
for example
using the following procedure. The target sequences and the nucleotide
location within the
'.5 HBV RNA are given in Tables 36-43.
Cleavage Reactions: Full-length or partially full-length, internally-labeled
target
RNA for ribozyme cleavage assay is prepared by in vitro transcription in the
presence of
[a-32p] CTP, passed over a G 50 Sephadex~ column by spin chromatography and
used as
substrate RNA without further purification. Alternately, substrates are 5'-32P-
end labeled
~0 using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming
a 2X
concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-
HCI, pH 7.5
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at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by adding
the 2X
ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was
also
pre-warmed in cleavage buffer. As an initial screen, assays are carried out
for 1 hour at
37°C using a final concentration of either 40 nM or 1 mM ribozyme,
i.e., ribozyme excess.
5 The reaction is quenched by the addition of an equal volume of 95%
formamide, 20 mM
EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is
heated to 95°C for 2 minutes, quick chilled and loaded onto a
denaturing polyacrylamide
gel. Substrate RNA and the specific RNA cleavage products generated by
ribozyme
cleavage are visualized on an autoradiograph of the gel. The percentage of
cleavage is
0 determined by Phosphor Imagei~ quantitation of bands representing the intact
substrate
and the cleavage products.
Transfection of HepG2 Cells with psHBV-1 and Ribozymes
The human hepatocellular carcinoma cell line Hep G2 was grown in Dulbecco's
5 modified Eagle media supplemented with 10% fetal calf serum, 2 mM glutamine,
0.1 mM
nonessential amino acids, 1 mM sodium pyruvate, 25 mM Hepes, 100 units
penicillin, and
100 ~g/ml streptomycin. To generate a replication competent cDNA, prior to
transfection
the HBV genomic sequences are excised from the bacterial plasmid sequence
contained in
the psHBV-1 vector (Those skilled in the art understand that other methods may
be used
'.0 to generate a replication competent cDNA). This was done with an EcoRI and
Hind III
restriction digest. Following completion of the digest, a ligation was
performed under
dilute conditions (20 pg/ml) to favor intermolecular ligation. The total
ligation mixture
was then concentrated using Qiagen spin columns.
Secreted alkaline phosphatase (SEAP) was used to normalize the HBsAg levels to
'S control for transfection variability. The pSEAP2-TK control vector was
constructed by
ligating a Bgl II-Hind III fragment of the pRL-TK vector (Promega), containing
the herpes
simplex virus thymidine kinase promoter region, into Bgl IIlHind III digested
pSEAP2-
Basic (Clontech). Hep G2 cells were plated (3 x 104 cells/well) in 96-well
microtiter plates
and incubated overnight. A lipid/DNA/ribozyme complex was formed containing
(at final
concentrations) cationic lipid (15 pg/ml), prepared psHBV-1 (4.5 pg/ml),
pSEAP2-TK
(0.5 pg/ml), and ribozyme (100 ~M). Following a 15 min. incubation at
37° C, the
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complexes were added to the plated Hep G2 cells. Media was removed from the
cells 96
hr. post-transfection for HBsAg and SEAP analysis.
Transfection of the human hepatocellular carcinoma cell line, Hep G2, with
replication competent HBV DNA results in the expression of HBV proteins and
the
production of virions. To investigate the potential use of ribozymes for the
treatment of
chronic HBV infection, a series of ribozymes that target the 3' terminus of
the HBV
genome have been synthesized. Ribozymes targeting this region have the
potential to
cleave all four major HBV RNA transcripts as well as the potential to block
the production
of HBV DNA by cleavage of the pregenomic RNA. To test the efficacy of these
HBV
ribozymes, they were co-transfected with HBV genomic DNA into Hep G2 cells,
and the
subsequent levels of secreted HBV surface antigen (HBsAg) were analyzed by
ELISA. To
control for variability in transfection efficiency, a control vector which
expresses secreted
alkaline phosphatase (SEAP), was also co-transfected. The efficacy of the HBV
ribozymes was determined by comparing the ratio of HBsAg:SEAP and/or
HBeAg:SEAP
to that of a scrambled attenuated control (SAC) ribozyme. Twenty-five
ribozymes
(RPI18341, RPI18356, RPI18363, RPI18364, RPI18365, RPI18366, RPI18367,
RPI18368,
RPI18369, RPI18370, RPI18371, RPI18372, RPI18373, RPI18374, RPI18303,
RPI18405,
RPI18406, RPI18407, RPI18408, RPI18409, RPI18410, RPI18411, RPI18418,
RPI18419,
and RPI18422) have been identified which cause a reduction in the levels of
HBsAg
and/or HBeAg as compared to the corresponding SAC ribozyme.
Example 6: Analysis of HBsA~ and SEAP Levels Following Ribozyme Treatment
Itnmulon 4 (Dynax) microtiter wells were coated overnight at 4° C with
anti-HBsAg
Mab (Biostride B88-95-3lad,ay) at 1 pg/ml in Carbonate Buffer (Na2C03 15 mM,
NaHC03 35 mM, pH 9.5). The wells were then washed 4x with PBST (PBS, 0.05%
Tween~ 20) and blocked for 1 hr at 37° C with PBST, 1% BSA. Following
washing as
above, the wells were dried at 37° C for 30 min. Biotinylated goat ant-
HBsAg (Accurate
YVS1807) was diluted 1:1000 in PBST and incubated in the wells for 1 hr. at
37° C. The
wells were washed 4x with PBST. Streptavidin/Alkaline Phosphatase Conjugate
(Pierce
21324) was diluted to 250 ng/ml in PBST, and incubated in the wells for 1 hr.
at 37° C.
After washing as above, p-nitrophenyl phosphate substrate (Pierce 37620) was
added to
the wells, which were then incubated for 1 hr. at 37° C. The optical
density at 405 nm was
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then determined. SEAP levels were assayed using the Great EscAPe~ Detection
Kit
(Clontech K2041-1), as per the manufacturers instructions.
Example 7: X- exporter Assay
The effect of ribozyme treatment on the level of transactivation of a SV40
promoter
driven firefly luciferase gene by the HBV X-protein was analyzed in
transfected Hep G2
cells. As a control for variability in transfection efficiency, a Renilla
luciferase reporter
driven by the TK promoter, which is not transactivated by the X protein, was
used. Hep
G2 cells were plated (3 x 104 cells/well) in 96-well microtiter plates and
incubated
overnight. A lipid/DNA/ribozyme complex was formed containing (at final
concentrations) cationic lipid (2.4 ~g/ml), the X-gene vector pSBDR(2.5
p,g/ml), the firefly
reporter pSV40HCVluc (0.5 pg/ml), the Renilla luciferase control vector pRL-TK
(0.5
pg/ml), and ribozyme (100 pM). Following a 15 min. incubation at 37° C,
the complexes
were added to the plated Hep G2 cells. Levels of firefly and Renilla
luciferase were
analyzed 48 hr. post transfection, using Promega's Dual-Luciferase Assay
System.
The HBV X protein is a transactivator of a number of viral and cellular genes.
Ribozymes which target the X region were tested for their ability to cause a
reduction in X
protein transactivation of a firefly luciferase gene driven by the SV40
promoter in
transfected Hep G2 cells. As a control for transfection variability, a vector
containing the
Renilla luciferase gene driven by the TK promotor, which is not activated by
the X protein,
was included in the co-transfections. The efficacy of the HBV ribozymes was
determined
by comparing the ratio of firefly luciferase: Renilla luciferase to that of a
scrambled
attenuated control (SAC) ribozyme. Eleven ribozymes (RPI18365, RPI18367,
RPI18368,
RPI18371, RPI18372, RPI18373, RPI18405; RPI18406, RPI18411, RPI18418,
RPI18423)
were identified which cause a reduction in the level of transactivation of a
reporter gene by
the X protein, as compared to the corresponding SAC ribozyme.
Example 8: HBV trans~enic mouse study
A transgenic mouse strain (founder strain 1.3.32 with a C57B1/6 background)
that
expresses HBV RNA and forms HBV viremia (Money et al., 1999, Antiviral Res.,
42, 97-
108; Guidotti et al., 1995, J. Virology, 69, 10, 6158-6169) was utilized to
study the in vivo
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activity of ribozymes of the instant invention. This model is predictive in
screening for
anti-HBV agents. Ribozyme or the equivalent volume of saline was administered
via a
continuous s.c. infusion using Alzet~ mini-osmotic pumps for 14 days. Alzet~
pumps
were filled with test materials) in a sterile fashion according to the
manufacturer's
instructions. Prior to in vivo implantation, pumps were incubated at
37°C overnight (> 18
hours) to prime the flow modulators. On the day of surgery, animals were
lightly
anesthetized with a ketamine/xylazine cocktail (94 mg/kg and 6 mg/kg,
respectively; 0.3
ml, 1P). Baseline blood samples (200 p1) were obtained from each animal via a
retro-
orbital bleed. A 2 cm area near the base of the tail was shaved and cleansed
with betadine
0 surgical scrub and sequentially with 70% alcohol. A 1 cm incision in the
skin was made
with a #15 scalpel blade or a blunt pair of scissors near the base of the
tail. Forceps were
used to open a pocket rostrally (i.e., towards the head) by spreading apart
the subcutaneous
connective tissue. The pump was inserted with the delivery portal pointing
away from the
incision. Wounds were closed with sterile 9-mm stainless steel clips or with
sterile 4-0
5 suture. Animals were then allowed to recover from anesthesia on a warm
heating pad
before being returned to their cage. Wounds were checked daily. Clips or
sutures were
replaced as needed. Incisions typically healed completely within 7 days post-
op. Animals
were then deeply anesthetized with the ketamine/xylazine cocktail (150 mg/kg
and 10
mg/kg, respectively; 0.5 ml, IP) on day 14 post pump implantation. A midline
'.0 thoracotomy/ laparatomy was performed to expose the abdominal cavity and
the thoracic
cavity. The left ventricle was cannulated at the base and animals
exsanguinated using a
23G needle and 1 ml syringe. Serum was separated, frozen and analyzed for HBV
DNA
and antigen levels. Experimental groups were compared to the saline control
group in
respect to percent change from day 0 to day 14. HBV DNA was assayed by
quantitative
'S PCR
Results
Table 44 is a summary of the group designation and dosage levels used in the
HBV
transgenic mouse study. Baseline blood samples were obtained via a
retroorbital bleed and
.0 animals (N=10/group) received anti-HBV ribozymes (100 mg/kg/day) as a
continuous SC
infusion. After 14 days, animals treated with a ribozyme targeting site 273
(RPL18341) of
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the HBV RNA showed a significant reduction in serum HBV DNA concentration,
compared to the saline treated animals as measured by a quantitative PCR
assay. More
specifically, the saline treated animals had a 69% increase in serum HBV DNA
concentrations over this 2-week period while treatment with the 273 ribozyme
(RPL18341) resulted in a 60% decrease in serum HBV DNA concentrations.
Ribozymes
directed against sites 1833 (RPI.18371), 1873 (RPI.18418), and 1874
(RPI.18372)
decreased serum HBV DNA concentrations by 49%, 15% and 16%, respectively.
Example 7: Activity of NCH Ribozyme to inhibit HER2 e~ ne expression
HER2 (also known as neu, erbB2 and c-erbB2) is an oncogene that encodes a 185-
kDa transmembrane tyrosine kinase receptor. HER2 is a member of the epidermal
growth
factor receptor (EGFR) family and shares partial homology with other family
members. In
normal adult tissues HER2 expression is low. However, HER2 is overexpressed in
at least
25-30% of breast (McGuire & Greene, 1989) and ovarian cancers (Berchuck, et
al., 1990).
5 Furthermore, overexpression of HER2 in malignant breast tumors has been
correlated with
increased metastasis, chemoresistance and poor survival rates (Slamon et al.,
1987 Science
235: 177-182). Because HER2 expression is high in aggressive human breast and
ovarian
cancers, but low in normal adult tissues, it is an attractive target for
ribozyme-mediated
therapy (Thompson et al., supra).
!0 The greatest HER2 specific effects have been observed in cancer cell lines
that
express high levels of HER2 protein (as measured by ELISA). Specifically, in
one study
that treated five human breast cancer cell lines with the HER2 antibody (anti-
erbB2-sFv),
the greatest inhibition of cell growth was seen in three cell lines (MDA-MB-
361, SKBR-3
and BT-474) that express high levels of HER2 protein. No inhibition of cell
growth was
!5 observed in two cell lines (MDA-MB-231 and MCF-7) that express low levels
of HERZ
protein (Wright et al., 1997). Another group successfully used SKBR-3 cells to
show
HER2 antisense oligonucleotide-mediated inhibition of HER2 protein expression
and
HER2 RNA knockdown (Vaughn et al., 1995). Other groups have also demonstrated
a
decrease in the levels of HER2 protein, HERZ mRNA and/or cell proliferation in
cultured
.0 cells using anti-HER2 ribozymes or antisense molecules (Suzuki, T. et al.,
1997; Weichen,
et al., 1997; Czubayko, F. et al., 1997; Colomer, et al., 1994; Betram et al.,
1994).
Because cell lines that express higher levels of HER2 have been more sensitive
to anti-
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HER2 agents, we are pursuing several medium to high expressing cell lines,
including
SKBR-3 and T47D, for ribozyme screens in cell culture.
A variety of endpoints have been used in cell culture models to look at HER2-
mediated effects after treatment with anti-HER2 agents. Phenotypic endpoints
include
inhibition of cell proliferation, apoptosis assays and reduction of HER2
protein expression.
Because overexpression of HER2 is directly associated with increased
proliferation of
breast and ovarian tumor cells, a proliferation endpoint for cell culture
assays will be our
primary screen. There are several methods by which this endpoint can be
measured.
Following treatment of cells with ribozymes, cells are allowed to grow
(typically 5 days)
0 after which either the cell viability, the incorporation of [3H] thymidine
into cellular DNA
and/or the cell density can be measured. The assay of cell density is very
straightforward
and can be done in a 96-well format using commercially available fluorescent
nucleic acid
stains (such as Syto 13 or CyQuant). The assay using CyQuant is in place at
RPI and is
currently being employed to screen 100 ribozymes targeting HER2 (details
below).
5 As a secondary, confirmatory endpoint a ribozyme-mediated decrease in the
level of
HER2 protein expression can be evaluated using a HER2-specific ELISA.
Validation of Cell Lines and Ribozyme Treatment Conditions
Two human breast cancer cell lines (T47D and SKBR-3) that are known to express
;0 medium to high levels of HER2 protein, respectively, were considered for
ribozyme
screening. In order to validate these cell lines for HER2-mediated
sensitivity, both cell
lines were treated with the HER2 specific antibody, Herceptin~ (Genentech) and
its effect
on cell proliferation was determined. Herceptin was added to cells at
concentrations
ranging from 0-8 ~,M in medium containing either no serum (OptiMem), 0.1% or
0.5%
'5 FBS and efficacy was determined via cell proliferation. Maximal inhibition
of
proliferation (~50%) in both cell lines was observed after addition of
Herceptin at 0.5 nM
in medium containing 0.1 % or no FBS. The fact that both cell lines are
sensitive to an
anti-HER2 agent (Herceptin) supports their use in experiments testing anti-
HER2
ribozymes.
.0 Prior to ribozyme screening, the choice of the optimal lipids) and
conditions for
ribozyme delivery was determined empirically for each cell line. Applicant has
established
a panel of proprietary lipids that can be used to deliver ribozymes to
cultured cells and are
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101
very usefizl for cell proliferation assays that are typically 3-5 days in
length. Initially, this
panel of proprietary lipid delivery vehicles was screened in SKBR-3 and T47D
cells using
previously established control oligonucleotides. Specific lipids and
conditions for optimal
delivery were selected for each cell line based on these screens. These
conditions were
used to deliver HER2 specific ribozymes to cells for primary (inhibition of
cell
proliferation) and secondary (decrease in HER2 protein) efficacy endpoints.
Primar~Screen: Inhibition of Cell Proliferation
Although optimal ribozyme delivery conditions were determined for two cell
lines,
I 0 the SKBR-3 cell line were be used for the initial screen because it has
the higher level of
HER2 protein, a.nd thus should be most susceptible to a HER2-specific
ribozyme. Follow-
up studies can be carned out in T47D cells to confirm leads as necessary.
Ribozyme screens were be performed using an automated, high throughput 96-well
cell proliferation assay. Cell proliferation were measured over a S-day
treatment period
5 using the nucleic acid stain CyQuant for determining cell density. The
growth of cells
treated with ribozyme/lipid complexes were compared to both untreated cells
and to cells
treated with Scrambled-arm Attenuated core Controls (SAC; or IA; Figure 8).
SACs can
no longer bind to the target site due to the scrambled arm sequence and have
nucleotide
changes in the core that greatly diminish ribozyme cleavage. These SACS are
used to
!0 determine non-specific inhibition of cell growth caused by ribozyme
chemistry (i.e.
multiple 2' Q-Me modified nucleotides, a single 2'C-allyl uridine, 4
phosphorothioates
and a 3' inverted abasic). Lead ribozymes are chosen from the primary screen
based on
their ability to inhibit cell proliferation in a specific manner. Dose
response assays are
carned out on these leads and a subset was advanced into a secondary screen
using the
!5 level of HER2 protein as an endpoint.
Secondary Screen: Decrease in HER2 Protein
A secondary screen that measures the effect of anti-HER2 ribozymes on HER2
protein levels is used to support preliminary findings. A robust HER2 ELISA
for both
.0 T47D and SKBR-3 cells has been established and is available for use as an
additional
endpoint.
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Ribozvme Mechanism Assays
A Taqman assay for measuring the ribozyme-mediated decrease in HER2 RNA has
also been established. :This assay is based on PCR technology and can measure
in real
time the production of HER2 mRNA relative to a standard cellular mRNA such as
GAPDH. This RNA assay is used to establish proof that lead ribozymes are
working
through an RNA cleavage mechanism and result in a decrease in the level of
HER2
mRNA, thus leading to a decrease in cell surface HER2 protein receptors and a
subsequent
decrease in tumor cell proliferation.
0 Animal Models
Evaluating the efficacy of anti-HER2 agents in animal models is an important
prerequisite to human clinical trials. As in cell culture models, the most
HER2 sensitive
mouse tumor xenografts are those derived from human breast carcinoma cells
that express
high levels of HER2 protein. In a recent study, nude mice bearing BT-474
xenografts
5 were sensitive to the anti-HER2 humanized monoclonal antibody Herceptin,
resulting in
an 80% inhibition of tumor growth at a 1 mg kg dose (ip, 2 X week for 4-5
weeks). Tumor
eradication was observed in 3 of 8 mice treated in this manner (Baselga et
al., 1998). This
same study compared the efficacy of Herceptin alone or in combination with the
commonly used chemotherapeutics, paclitaxel or doxorubicin. Although, all
three anti-
'.0 HER2 agents caused modest inhibition of tumor growth, the greatest
antitumor activity
was produced by the combination of Herceptin and paclitaxel (93% inhibition of
tumor
growth vs 35% with paclitaxel alone). The above studies provide proof that
inhibition of
HER2 expression by anti-HER2 agents causes inhibition of tumor growth in
animals.
Lead anti-HER2 ribozymes chosen from in vitro assays are further tested in
mouse
'S xenograft models. Ribozymes are first tested alone and then in combination
with standard
chemotherapies.
Animal Model Development
Three human breast tumor cell lines (T47D, SKBR-3 and BT-474) were
characterized to establish their growth curves in mice. These three cell lines
have been
implanted into the mammary papillae of both nude and SC>I7 mice and primary
tumor
volumes are measured 3 times per week. Growth characteristics of these tumor
lines using
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a Matrigel implantation format will also be established. In addition, the use
of two other
breast cell lines that have been engineered to express high levels of HER2 are
also being
used. The tumor cell lines) and implantation method that supports the most
consistent
and reliable tumor growth is used in animal studies testing the lead HER2
ribozyme(s).
Ribozyme are administered by daily subcutaneous injection or by continuous
subcutaneous
infusion from Alzet mini osmotic pumps beginning 3 days after tumor
implantation and
continuing for the duration of the study. Group sizes of at least 10 animals
are employed.
Efficacy is determined by statistical comparison of tumor volume of ribozyme-
treated
animals to a control group of animals treated with saline alone. Because the
growth of
0 these tumors is generally slow (45-60 days), an initial endpoint will be the
time in days it
takes to establish an easily measurable primary tumor (i.e. SO-100 mm3) in the
presence or
absence of ribozyme treatment.
Clinical Summary
5 Breast cancer is a common cancer in women and also occurs in men to a lesser
degree. The incidence of breast cancer in the United States is 180,000 cases
per year and
46,000 die each year of the disease. In addition, 21,000 new cases of ovarian
cancer per
year lead to 13,000 deaths (data from Hung et al., 1995 and the Surveillance,
Epidemiology and End Results Program, NCI). Ovarian cancer is a potential
secondary
'0 indication for anti-HER2 ribozyme therapy.
A full review of breast cancer is given in the NCI PDQ for Breast Cancer. A
brief
overview is given here. Breast cancer is evaluated or "staged" on the basis of
tumor size,
and whether it has spread to lymph nodes and/or other parts of the body. In
Stage I breast
cancer, the cancer is no larger than 2 centimeters and has not spread outside
of the breast.
5 In Stage II, the patient's tumor is 2-5 centimeters but cancer may have
spread to the
axillary lymph nodes. By Stage III, metastasis to the lymph nodes is typical,
and tumors
are S centimeters. Additional tissue involvement (skin, chest wall, ribs,
muscles etc.)
may also be noted. Once cancer has spread to additional organs of the body, it
is classed
as Stage IV.
0 Almost all breast cancers (>90%) are detected at Stage I or II, but 31% of
these are
already lymph node positive. The S-year survival rate for node negative
patients (with
standard surgery/radiation/chemotherapy /hormone regimens) is 97%; however,
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involvement of the lymph nodes reduces the 5-year survival to only 77%.
Involvement of
other organs ( Stage III) drastically reduces the overall survival, to 22% at
5 years. Thus,
chance of recovery from breast cancer is highly dependent on early detection.
Because up
to 10% of breast cancers are hereditary, those with a family history are
considered to be at
high risk for breast cancer and should be monitored very closely.
Breast cancer is highly treatable and often curable when detected in the early
stages.
(For a complete review of breast cancer treatments, see the NCI PDQ for Breast
Cancer.)
Common therapies include surgery, radiation therapy, chemotherapy and hormonal
therapy. Depending upon many factors, including the tumor size, lymph node
involvement
0 and location of the lesion, surgical removal varies from lumpectomy (removal
of the tumor
and some surrounding tissue) to mastectomy (removal of the breast, lymph nodes
and
some or all of the underlying chest muscle). Even with successful surgical
resection, as
many as 21% of the patients may ultimately relapse (10-20 years). Thus, once
local
disease is controlled by surgery, adjuvant radiation treatments,
chemotherapies and/or
5 hormonal therapies are typically used to reduce the rate of recurrence and
improve
survival. The therapy regimen employed depends not only on the stage of the
cancer at its
time of removal, but other variables such the type of cancer (ductal or
lobular), whether
lymph nodes were involved and removed, age and general health of the patient
and if other
organs are involved.
'.0 Common chemotherapies include various combinations cytotoxic drugs to kill
the
cancer cells. These drugs include paclitaxel (Taxol), docetaxel, cisplatin,
methotrexate,
cyclophosphamide, doxorubin, fluorouracil etc. Significant toxicities are
associated with
these cytotoxic therapies. Well-characterized toxicities include nausea and
vomiting,
myelosuppression, alopecia and mucosity. Serious cardiac problems are also
associated
'.5 with certain of the combinations, e.g. doxorubin and paclitaxel, but are
less common.
Testing for estrogen and progesterone receptors helps to determine whether
certain
anti-hormone therapies might be helpful in inhibiting tumor growth. If either
or both
receptors are present, therapies to interfere with the action of the hormone
ligands, can be
given in combination with chemotherapy and are generally continued for several
years.
~0 These adjuvant therapies are called SERMs, selective estrogen receptor
modulators, and
they can give beneficial estrogen-like effects on bone and lipid metabolism
while
antagonizing estrogen in reproductive tissues. Tamoxifen is one such compound.
The
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primary toxic effect associated with the use of tamoxifen is a 2 to 7-fold
increase in the
rate of endometrial cancer. Blood clots in the legs and lung and the
possibility of stroke
are additional side effects. However, tamoxifen has been determined to reduce
breast
cancer incidence by 49% in high-risk patients and an extensive, somewhat
controversial,
clinical study is underway to expand the prophylactic use of tamoxifen.
Another SERM,
raloxifene, was also shown to reduce the incidence of breast cancer in a large
clinical trial
where it was being used to treat osteoporosis. In additional studies, removal
of the ovaries
and/or drugs to keep the ovaries from working are being tested.
Bone marrow transplantation is being studied in clinical trials for breast
cancers that
0 have become resistant to traditional chemotherapies or where >3 lymph nodes
are
involved. Marrow is removed from the patient prior to high-dose chemotherapy
to protect
it from being destroyed, and then replaced after the chemotherapy. Another
type of
"transplant" involves the exogenous treatment of peripheral blood stem cells
with drugs to
kill cancer cells prior to replacing the treated cells in the bloodstream.
5 One biological treatment, a humanized monoclonal anti-HER2 antibody,
Herceptin
(Genentech) has been approved by the FDA as an additional treatment for HER2
positive
tumors. Herceptin binds with high affinity to the extracellular domain of HER2
and thus
blocks its signaling action. Herceptin can be used alone or in combination
with
chemotherapeutics (i.e. paclitaxel, docetaxel, cisplatin, etc.) (Pegram, et
al., 1998). In
0 Phase III studies, Herceptin significantly improved the response rate to
chemotherapy as
well as improving the time to progression (Ross & Fletcher, 1998). The most
common
side effects attributed to Herceptin are fever and chills, pain, asthenia,
nausea, vomiting,
increased cough, diarrhea, headache, dyspnea, infection, rhinitis, and
insomnia. Herceptin
in combination with chemotherapy (paclitaxel) can lead to cardiotoxicity
(Sparano, 1999),
5 leukopenia, anemia, diarrhea, abdominal pain and infection.
HER2 Protein Levels for Patient Screenine and as a Potential Endpoint
Because elevated HER2 levels can be detected in at least 30% of breast
cancers,
breast cancer patients can be pre-screened for elevated HER2 prior to
admission to initial
0 clinical trials testing an anti-HERZ ribozyme. Initial HER2 levels can be
deterniined (by
ELISA) from tumor biopsies or resected tumor samples.
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During clinical trials, it may be possible to monitor circulating HER2 protein
by
ELISA (Ross and Fletcher, 1998). Evaluation of serial blood/serum samples over
the
course of the anti-HER2 ribozyme treatment period could be useful in
determining early
indications of efficacy. In fact, the clinical course of Stage IV breast
cancer was correlated
with shed HER2 protein fragment following a dose-intensified paclitaxel
monotherapy. In
all responders, the HER2 serum level decreased below the detection limit
(Luftner et al.).
Two cancer-associated antigens, CA27.29 and CA15.3, can also be measured in
the
serum. Both of these glycoproteins have been used as diagnostic markers for
breast
cancer. CA27.29 levels are higher than CA15.3 in breast cancer patients; the
reverse is
I 0 true in healthy individuals. Of these two markers, CA27.29 was found to
better
discriminate primary cancer from healthy subjects. In addition, a
statistically significant
and direct relationship was shown between CA27.29 and large vs small tumors
and node
postive vs node negative disease (Gion, et al., 1999). Moreover, both cancer
antigens were
found to be suitable for the detection of possible metastases during follow-up
(Rodriguez
I 5 de Paterna et al., 1999). Thus, blocking breast tumor growth may be
reflected in lower
CA27.29 and/or CA15.3 levels compared to a control group. FDA submissions for
the use
of CA27.29 and CA15.3 for monitoring metastatic breast cancer patients have
been filed
(reviewed in Beveridge, 1999). Fully automated methods for measurement of
either of
these markers are commercially available.
!0
References
Baselga, J., Norton, L. Albanell, J., Kim, Y.M. and Mendelsohn, J. (1998)
Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor
activity
of paclitaxel and doxorubicin against HER2/neu overexpressing human breast
cancer
!5 xenografts. Cancer Res. 15: 2825-2831.
Berchuck, A. Kamel, A., Whitaker, R. et al. (1990) Overexpression of her-2/neu
is
associated with poor survival in advanced epithelial ovarian cancer. Cancer
Research S0:
4087-4091.
Bertram, J. Killian, M., Brysch, W., Schlingensiepen, K.-H., and Kneba, M.
(1994)
.0 Reduction of erbB2 gene product in mamma carcinoma cell lines by erbB2 mRNA-
specific and tyrosine kinase consensus phosphorothioate antisense
oligonucleotides.
Biochem. BioPhys. Res. Comm. 200: 661-667.
CA 02403243 2002-02-21
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107
Beveridge, R.A. (1999) Review of clinical studies of CA27.29 in breast cancer
management. Int. J. Biol. Markers 14: 36-39.
Colomer, R., Lupu, R., Bacus, S.S. and Gelmann, E.P. (1994) erbB-2 antisense
oligonucloetides inhibit the proliferation of breast carcinoma cells with erbB-
2 oncogene
amplification. British J. Cancer 70: 819-825.
Czubayko, F., Downing, S.G., Hsieh, S.S., Goldstein, D.J., Lu P.Y., Trapnell,
B.C.
and Wellstein, A. (1997) Adenovirus-mediated transduction of ribozymes
abrogates HER-
2/neu and pleiotrophin expression and inhibits tumor cell proliferation. Gene
Ther. 4:
943-949.
0 Gion, M., Mione, R., Leon, A.E. and Dittadi, R. (1999) Comparison of the
diagnostic accuracy of CA27.29 and CA15.3 in primary breast cancer. Clin.
Chem. 45:
630-637.
Hung, M.-C., Matin, A., Zhang, Y., Xing, X., Sorgi, F., Huang, L. and Yu, D.
(1995)
HER-2/neu-targeting gene therapy - a review. Gene 159: 65-71.
5 Luftner, D., Schnabel. S. and Possinger, K. (1999) c-erbB-2 in serum of
patients
receiving fractionated paclitaxel chemotherapy. Int. J. Biol. Markers 14: 55-
59.
McGuire, H.C. and Greene, M.I. (1989) The neu (c-erbB-2) oncogene. Semin.
Oncol. 16: 148-155.
NCI PDQ/Treatment/Health ProfessionalsBreast Cancer:
'0 http://cancernet.nci.nih.gov/clinpdq/soaBreast cancer Physician.html
NCI PDQ/Treatrnent/PatientsBreast Cancer:
httn://cancernet.nci.nih. ovg /clinpdq/pif/Breast cancer Patient.html
Pegram, M.D., Lipton, A., Hayes, D.F., Weber, B.L., Baselga, J.M., Tripathy,
D.,
Baly, D., Baughman, S.A., Twaddell, T., Glaspy, J.A. and Slamon, D.J. (1998)
Phase II
5 study of receptor-enhanced chemosensitivity using recombinant humanized anti-
p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-
overexpressing metastatic breast cancer refractory to chemotherapy treatment.
J. Clin.
Oncol. 16: 2659-2671.
Rodriguez de Paterna, L., Arnaiz, F., Estenoz, J. Ortuno, B. and Lanzos E.
(1999)
0 Study of serum tumor markers CEA, CA15.3, CA27.29 as diagnostic parameters
in
patients with breast carcinoma. Int. J. Biol. Markers 10: 24-29.
CA 02403243 2002-02-21
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108
Ross, J.S. and Fletcher, J.A. (1998) The HER-2/neu oncogene in breast cancer:
Prognostic factor, predictive factor and target for therapy. Oncologist 3:
1998.
Slamon, D.J., Clark, G.M., Wong, S.G., Levin, W.J., Ullrich, A. and McGuire,
W.L.
(1987) Human breast cancer: correlation of relapse and survival with
amplification of the
HER-2/neu oncogene. Science 235: 177-182.
Sparano, J.A. (1999) Doxorubicin/taxane combinations: Cardiac toxicity and
pharmacokinetics. Semin. Oncol. 26: 14-19.
Surveillance, Epidemiology and End Results Program (SEER) Cancer Statistics
Review: http://www.seer.ims.nci.nih.gov/Publications/CSR1973_1996/
0 Suzuki T., Curcio, L.D., Tsai, J. and Kashani-Sabet M. (1997) Anti-c-erb-B-2
Ribozyme for Breast Cancer. In Methods in Molecular Medicine, Vol. 11,
Therapeutic
Applications of Ribozmes, Human Press, Inc., Totowa, NJ.
aughn, J.P., Iglehart, J.D., Demirdji, S., Davis, P., Babiss, L.E., Caruthers,
M.H.,
Marks, J.R. (1995) Antisense DNA downregulation of the ERBB2 oncogene measured
by
5 a flow cytometric assay. Proc Natl Acad Sci USA 92: 8338-8342.
Weichen, K., Zimmer, C. and Dietel, M. (1997) Selection of a high activity c-
erbB-2
ribozyme using a fusion gene of c-erbB-2 and the enhanced green fluorescent
protein.
Cancer Gene Therapy S: 45-51.
Wright, M., Grim, J., Deshane, J., Kim, M., Strong, T.V., Siegel, G.P.,
Curiel, D.T.
0 (1997) An intracellular anti-erbB-2 single-chain antibody is specifically
cytotoxic to
human breast carcinoma cells overexpressing erbB-2. Gene Therapy 4: 317-322.
Applicant has designed, synthesized and tested several NCH ribozymes and HH
ribozymes targeted against HER2 RNA (see for example Tables 31 and 34) in cell
proliferation assays.
5
Proliferation assay:
The model proliferation assay used in the study can require a cell plating
density of
2000 cells/well in 96-well plates and at least 2 cell doublings over a 5-day
treatment
period. To calculate cell density for proliferation assays, the FIPS (fluoro-
imaging
0 processing system) method well in the art was used. This method allows for
cell density
measurements after nucleic acids are stained with CyQuant dye, and has the
advantage of
CA 02403243 2002-02-21
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109
accurately measuring cell densities over a very wide range 1,000-100,000
cells/well in 96-
well format.
Ribozymes (50-200 nM) were delivered in the presence of cationic lipid at 2.0
pg/mL and inhibition of proliferation was determined on day 5 post-treatment.
Two full
ribozyme screens were completed and 4 lead HH and 11 lead NCH ribozymes were
chosen
for further testing. Of the 15 lead Rzs chosen from primary screens, 4 NCH and
1 HH
Rzs continued to inhibit cell proliferation in subsequent experiments. NCH Rzs
against
sites, 2001 (RPI No. 17236), 2783 (RPI No. 17249), 2939 (RPI No. 17251) or
3998 (RPI
No. 17262) caused inhibition of proliferation ranging from 25-60% as compared
to a
I 0 scrambled control Rz (IA; RPI No. 17263). Of the five lead Rzs, the most
efficacious is
the NCH Rz (RPI No. 17251) against site 2939 of HER2 RNA. An example of
results
from cell culture assay is shown in Figure 8. Referring to Figure 8, NCH
ribozymes and
a HH ribozyme targeted against HER2 RNA, are shown to cause significant
inhibition of
proliferation of cells. This shows that ribozymes, for instance the NCH
ribozymes are
5 capable of inhibiting HER2 gene expression in mammalian cells.
Example 8: Activity of Class II (Zinzvme) nucleic acid catalysts to inhibit
HER2 gene
e~ression
Applicant has designed, synthesized and tested several class II (zinzyme)
ribozymes
!0 targeted against HER2 RNA (see, for example, Tables 58, 59, and 62) in cell
proliferation
RNA reduction assays.
Proliferation assay:
The model proliferation assay used in the study requires a cell-plating
density of
'.5 2000-10000 cells/well in 96-well plates and at least 2 cell doublings over
a 5-day treatment
period. Cells used in proliferation studies were either human breast or
ovarian cancer cells
(SKBR-3 and SKOV-3 cells respectively). To calculate cell density for
proliferation
assays, the FIPS (fluoro-imaging processing system) method well known in the
art was
used. This method allows for cell density measurements after nucleic acids are
stained
~0 with CyQuant~ dye, and has the advantage of accurately measuring cell
densities over a
very wide range 1,000-100,000 cells/well in 96-well format.
CA 02403243 2002-02-21
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110
Ribozymes (50-200 nM) were delivered in the presence of cationic lipid at 2.0-
5.0
pg/mL and inhibition of proliferation was determined on day 5 post-treatment.
Two full
ribozyme screens were completed resulting in the selection of 14 ribozymes.
Class II
(zinzyme) ribozymes against sites, 314 (RPI No. 18653), 443 (RPI No. 18680),
597 (RPI
No. 18697), 659 (RPI No. 18682), 878 (RPI Nos. 18683 and 18654), 881 (RPI Nos.
18684
and 18685) 934 (RPI No. 18651), 972 (RPI No. 18656, 19292, 19727, 19728, and
19293),
1292 (RPI No. 18726), 1541 (RPI No. 18687), 2116 (RPI No. 18729), 2932 (RPI
No.
18678), 2540 (RPI No. 18715), and 3504 (RPI No. 18710) caused inhibition of
proliferation ranging from 25-80% as compared to a scrambled control ribozyme.
An
0 example of results from a cell culture assay is shown in Figure 20.
Referring to Figure
20, Class II ribozymes targeted against HER2 RNA are shown to cause
significant
inhibition of proliferation of cells. This shows that ribozymes, for instance
the Class II
(zinzyme) ribozymes are capable of inhibiting HER2 gene expression in
mammalian cells.
5 RNA assay:
RNA was harvested 24 hours post-treatment using the Qiagen RNeasy~ 96
procedure. Real time RT-PCR (TaqMan~ assay) was performed on purified RNA
samples using separate primer/probe sets specific for either target HER2 RNA
or control
actin RNA (to normalize for differences due to cell plating or sample
recovery). Results
'.0 are shown as the average of triplicate determinations of HER2 to actin RNA
levels post-
treatment. Figure 30 shows class II ribozyme (zinzyme) mediated reduction in
HER2
RNA targeting site 972 vs a scrambled attenuated control.
Dose response assays:
.5 Active ribozyme was mixed with binding arm-attenuated control (BAC)
ribozyme to
a final oligonucleotide concentration of either 100, 200 or 400 nM and
delivered to cells in
the presence of cationic lipid at 5.0 pg/mL. Mixing active and BAC in this
manner
maintains the lipid to ribozyme charge ratio throughout the dose response
curve. HER2
RNA reduction was measured 24 hours post-treatment and inhibition of
proliferation was
determined on day 5 post-treatment. The dose response antiproliferation
results are
summarized in Figure 31 and the dose-dependent reduction of HER2 RNA results
are
CA 02403243 2002-02-21
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111
summarized in Figure 32. Figure 33 shows a combined dose response plot of both
anti-
proliferation and RNA reduction data for a class II ribozyme targeting site
972 of HER2
RNA (RPI 19293).
Example 9: Compositions having RNA cleavin. activity
Hammerhead ribozymes are an example of catalytic RNA molecules which are able
to recognize and cleave a given specific RNA substrate (Hutchins et a1.,1986,
Nucleic
Acids Res. 14:3627; Keese and Symons, in Yiroids and viroid-like pathogens
(J.J.
Semanchik, publ., CRC-Press, Boca Raton, Florida, 1987, pages 1-47). The
catalytic
0 center of hammerhead ribozymes is flanked by three stems and can be formed
by adjacent
sequence regions of the RNA or also by regions, which are separated from one
another by
many nucleotides. Figure 6 shows a diagram of such a catalytically active
hammerhead
structure. The stems have been denoted I, II and III. The nucleotides are
numbered
according to the standard nomenclature for hammerhead ribozymes (Hertel et
al., 1992,
5 Nucleic Acids Res. 20:3252). In this nomenclature, bases are denoted by a
number, which
relates their position relative to the S' side of the cleavage site.
Furthermore, each base that
is involved in a stem or loop region has an additional designation (which is
denoted by a
decimal point and then another number) that defines the position of that base
within the
stem or loop. A designation of A'S'' would indicate that this base is involved
in a paired
0 region and that it is the first nucleotide in that stem going away from the
core region. This
accepted convention for describing hammerhead-derived ribozymes allows for the
nucleotides involved in the core of the enzyme to always have the same number
relative to
all of the other nucleotides. The size of the stems involved in substrate
binding or core
formation can be any size and of any sequence, and the position of A9, for
example, will
5 remain the same relative to all of the other core nucleotides. Nucleotides
designated, for
example, N~'2 or N9~ represent an inserted nucleotide where the position of
the caret (~)
relative to the number denotes whether the insertion is before or after the
indicated
nucleotide. Thus, N~' 2 represents a nucleotide inserted before nucleotide
position 12, and
N9~ represents a nucleotide inserted after nucleotide position 9.
0 The consensus sequence of the catalytic core structure is described by
Ruffner and
LThlenbeck, 1990, Nucleic Acids Res. 18:6025-6029. Perriman et al., 1992, Gene
113:157-
163, have meanwhile shown that this structure can also contain variations, for
example,
CA 02403243 2002-02-21
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112
naturally occurring nucleotide insertions such as N9~ and N~'2. Thus, the
positive strand
of the satellite RNA of the tobacco ring-spot virus does not contain any of
the two
nucleotide insertions while the +RNA strand of the virusoid of the lucerne
transient streak
virus (vLTSV) contains a N9~ = U insertion which can be mutated to C or G
without loss
of activity (Sheldon and Symons, 1989, Nucleic Acids Res. 17:5679-5685).
Furthermore,
in this special case, N' = A and R's.' = A. On the other hand, the minus
strand of the
carnation stunt associated viroid (-CarSV) is quite unusual since it contains
both
nucleotide insertions, that is N~'2 = A and N9~ = C (Hernandez et a1.,1992,
Nucleic Acids
Res. 20:6323-6329). In this viroid N' = A and R's'' = A. In addition, this
special
0 hammerhead structure exhibits a very effective self catalytic cleavage
despite the more
open central stem.
Possible uses of hammerhead ribozymes include, for example, generation of RNA
restriction enzymes and the specific inactivation of the expression of genes
in, for
example, animal, human or plant cells and prokaryotes, yeasts and plasmodia. A
particular
5 biomedical interest is based on the fact that many diseases, including many
forms of
tumors, are related to the overexpression of specific genes. Inactivating such
genes by
cleaving the associated mRNA represents a possible way to control and
eventually treat
such diseases. Moreover there is a great need to develop antiviral,
antibacterial, and
antifungal pharmaceutical agents. Ribozymes have potential as such anti-
infective agents
'.0 since RNA molecules vital to the survival of the organism can be
selectively destroyed.
In addition to needing the correct hybridizing sequences for substrate
binding,
substrates for hammerhead ribozymes have been shown to strongly prefer the
triplet
Ni6.zUi6.iHm (~) where N can be any nucleotide, U is uridine, and H is either
adenosine, cytidine, or uridine (Koizumi et al., 1988, FEBS Lett. 228, 228-
230; Ruffner et
'.5 al., 1990, Biochemistry 29, 10695-10702 ; Pernman et al., 1992, Gene 113,
157-163).
NUH is sometimes designated as NUX. The fact that changes to this general rule
for
substrate specificity result in non-functional substrates implies that there
are "non core
compatible" structures which are formed when substrates are provided which
deviate from
the stated requirements. Evidence along these lines was recently reported by
Uhlenbeck
~0 and co-workers (Uhlenbeck et al., 1997, Biochemistry 36:1108-1114) when
they
demonstrated that the substitution of a G at position 17 caused a functionally
catastrophic
base pair between G" and C3 to form, both preventing the correct orientation
of the
CA 02403243 2002-02-21
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113
scissile bond for cleavage and the needed tertiary interactions of C3 (Murray
et al., 1995,
Biochem. J. 311:487-494). The strong preference for a U at position 16.1 may
exist for
similar reasons. Many experiments have been done in an attempt to isolate
ribozymes
which are able to efficiently relieve the requirement of a U at position 16.1,
however,
attempts to find hammerhead type ribozymes which can cleave substrates having
a base
other than a U at position 16.1 have proven impossible (Perriman et al., 1992,
Gene 113,
157-163).
Efficient catalytic molecules with reduced or altered requirements in the
cleavage
region are highly desirable because their isolation would greatly increase the
number of
I 0 available target sequences that molecules of this type could cleave. For
example, it would
be desirable to have a ribozyme variant that could efficiently cleave
substrates containing
triplets other than Nl6.zUi6.iHm since this would increase the number of
potential target
cleavage sites.
Chemically modified oligonucleotides which contain a block of
5 deoxyribonucleotides in the middle region of the molecule have potential as
pharmaceutical agents for the specific inactivation of the expression of genes
(Giles et al.,
1992, Nucleic Acids Res. 20:763-770). These oligonucleotides can form a hybrid
DNA-
RNA duplex in which the DNA bound RNA strand is degraded by RNase H. Such
oligonucleotides are considered to promote cleavage of the RNA and so cannot
be
!0 characterized as having an RNA-cleaving activity nor as cleaving an RNA
molecule (the
RNase H is cleaving). A significant disadvantage of these oligonucleotides for
in vivo
applications is their low specificity, since hybrid formation, and thus
cleavage, can also
take place at undesired positions on the RNA molecules.
Since, unmodified ribozymes are sensitive to degradation by RNases, chemically
!5 modified active substances have to be used in order to administer
hammerhead ribozymes
exogenously (discussed, for example, by Heidenreich et al., 1994, J. Biol.
Chem.
269:2131-2138; Kiehntopf et al., 1994, EMBO J. 13:4645-4652; Paolella et al.,
1992,
EMBO J. 11:1913-1919; and Usman et al., 1994, Nucleic Acids Symp. Ser. 31:163-
164).
Sproat et al., U.S. Pat. No. 5,334,711, describe such chemically modified
active
.0 substances based on synthetic catalytic oligonucleotide structures with a
length of 35 to 40
nucleotides which are suitable for cleaving a nucleic acid target sequence and
contain
modified nucleotides that contain an optionally substituted alkyl, alkenyl or
alkynyl group
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with 1 - 10 carbon atoms at the 2'-O atom of the ribose. These
oligonucleotides contain
modified nucleotide building blocks and form a structure resembling a
hammerhead
structure. These oligonucleotides are able to cleave specific RNA substrates.
Usman et al., U.S. Patent No. 5,891,684, describe enzymatic nucleic acid
molecules
with one or more nucleotide base modifications) in a substrate binding arm.
Thompson et al., US Patent No. 5,599,704 describe enzymatic RNA molecules
targeted against ErbB2/neu/Her2 RNA.
Sullivan et al., US Patent No. 5,616,490 describe enzymatic RNA molecules
targeted against protein kinase C (PKC) RNA.
0 Sioud, International PCT publication No. WO 99/63066 describe hammerhead
ribozymes targeted against specific sites within protein kinase C alpha (PKC
alpha),
VEGF, and TNF alpha RNA.
Jarvis et al., International PCT publication No. WO 98/505030, describe the
synthesis of xylo-ribonucleosides and oligonucleotides comprising xylo
modifications.
5 This invention relates to novel enzymatic nucleic acid molecules having an
RNA-
cleavage activity, as well as their use for cleaving RNA substrates in vitro
and in vivo. The
compositions contain an active center, the subunits of which are selected from
nucleotides
and/or nucleotide analogues, as well as flanking regions contributing to the
formation of a
specific hybridization with an RNA substrate. Preferred compositions form, in
0 combination with an RNA substrate, a structure resembling a hammerhead
structure. The
active center of the disclosed compositions is characterized by the presence
of h5-1 which
allows cleavage of RNA substrates having 016.1. It is therefore an object of
the present
invention to provide compositions that cleave RNA, and in particular to
provide RNA-
cleaving oligomers which at the same time have a high stability, activity, and
specificity.
5 This invention relates to novel nucleic acid molecules with catalytic
activity, which are
particularly useful for cleavage of RNA or DNA or combination thereof. The
nucleic acid
catalysts of the instant invention are distinct from other nucleic acid
catalysts known in the
art. Specifically, nucleic acid catalysts of the instant invention are capable
of catalyzing an
intermolecular or intramolecular endonuclease reaction.
0 It is another object of the present invention to provide compositions that
cleave RNA
substrates having a cleavage site triplet other than N'6.2U16.1Hm (~~ Figure
6), where N
is a nucleotide, U is uridine and H is adenosine, uridine or cytidine. H is
used
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interchangably with X. Specifically, the enzymatic nucleic acid molecule of
the instant
invention has an endonuclease activity to cleave RNA substrates having a
cleavage triplet
NI6.2C16.1H17 (NCH; Figure 6),. where N is a nucleotide, C is cytidine and H
is adenosine,
uridine or cytidine. H is used interchangeably with X. In another aspect the
invention
features an enzymatic nucleic acid molecule of the instant invention has an
endonuclease
activity to cleave RNA substrates having a cleavage triplet N16.2C16.1N17
(NCN; Figure 6),
where N is a nucleotide, C is cytidine.
In a preferred embodiment, the invention features an enzymatic nucleic acid
molecule having formula 1:
- G-A-A-I
L
\~)n-~)p A-G-N-A -G-U-C-E-5'
where N represents independently a nucleotide or a non-nucleotide linker,
which
may be same or different; D and E are independently oligonucleotides of length
sufficient
to stably interact (e.g., by forming hydrogen bonds with complementary
nucleotides in the
target) with a target nucleic acid molecule (the target can be an RNA, DNA or
mixed
5 polymers), preferably, the length of D and E are independently between 3-20
nucleotides
long, specifically, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 S, 17, and 20;
o and n are integers
independently greater than or equal to 1 and preferably less than about 100,
specifically 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 50, wherein if (I~o and (l~n are
nucleotides, (I~o
and (l~n are optionally able to interact by hydrogen bond interaction, in
particular if n =1
!0 and o=1 then (N)n is preferably a purine (e.g., G, and A) and (I~o is
preferably a
pyrimidine (e.g., C and U) and (I~n preferably forms; ~ indicates base-paired
interaction;
L is a linker which may be present or absent (i.e., the molecule may be
assembled from
two separate oligonucleotides), but when present, is a nucleotide and/or a non-
nucleotide
linker, which may be a single-stranded and/or double-stranded region; p is an
integer 0 or
!5 1, when p=l, (I~p is preferably A or U; and represents a chemical linkage
(e.g. a
phosphate ester linkage, amide linkage, phosphorothioate linkage or others
known in the
art). A, U, I, C and G represent adenosine, uridine, inosine, cytidine and
guanosine
nucleotides, respectively. The N in S'-CUGANGA-3' region of formula 1 is
preferably U.
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The nucleotides in the formula 1 are unmodified or modified at the sugar,
base, and/or
phosphate as known in the art.
In a preferred embodiment, the invention features an enzymatic nucleic acid
molecule having formula 2:
/ C G_ A_ A_ I D
L
~ CN) n G CN) p _a- G_ N_ A_ G- U- C _ E _ 5'
where N represents independently a nucleotide or a non-nucleotide linker,
which
may be same or different; D and E are independently oligonucleotides of length
sufficient
to stably interact (e.g., by forming hydrogen bonds with complementary
nucleotides in the
0 target) with a target nucleic acid molecule (the target can be an RNA, DNA
or mixed
polymers), preferably, the length of D and E are independently between 3-20
nucleotides
long, specifically, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, and 20; o
and n are integers
independently greater than or equal to 0 and preferably less than about 100,
specifically 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50, wherein if (I~o and (I~n are
nucleotides, (loo
5 and (N)n are optionally able to interact by hydrogen bond interaction; ~
indicates base-
paired interaction; L is a linker which may be present or absent (i. e., the
molecule may be
assembled from two separate oligonucleotides), but when present, is a
nucleotide and/or a
non-nucleotide linker, which may be a single-stranded and/or double-stranded
region; p is
an integer 0 or 1, when p=1, (I~p is preferably A, C or U; and represents a
0 chemical linkage (e.g. a phosphate ester linkage, amide linkage,
phosphorothioate linkage
or others known in the art). A, U, I, C and G represent adenosine, uridine,
inosine, cytidine
and guanosine nucleotides, respectively. The N in 5'-CUGANGA-3' region of
formula 2
is preferably U. The nucleotides in the formula 2 are unmodified or modified
at the sugar,
base, and/or phosphate as known in the art.
5 In a preferred embodiment, the I (inosine) in formula l and 2 is preferably
a ribo-
inosine or a xylo-inosine.
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In yet another embodiment, the nucleotide linker (L) is a nucleic acid
aptamer, such
as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others
(for a
review see Gold et al., 1995, Annu. Rev. Biochem., 64, 763; and Szostak &
Ellington,
1993, in The RNA World, ed. Gesteland and Atkins, pp 511, CSH Laboratory
Press). A
"nucleic acid aptamer" as used herein is meant to indicate nucleic acid
sequence capable of
interacting with a ligand. The ligand can be any natural or a synthetic
molecule, including
but not limited to a resin, metabolites, nucleosides, nucleotides, drugs,
toxins, transition
state analogs, peptides, lipids, proteins, amino acids, nucleic acid
molecules, hormones,
carbohydrates, receptors, cells, viruses, bacteria and others. In a preferred
embodiment L
0 has the sequence 5'-GAAA-3' or 5'-GUUA-3'.
In yet another embodiment, the non-nucleotide linker (L) is as defined herein.
The term "non-nucleotide", as used herein, includes either abasic nucleotide,
polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or
polyhydrocarbon
compounds. Specific examples include those described by Seela and Kaiser,
Nucleic
5 Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and
Schepartz, J.
Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc.
1991,
113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993,
32:1751;
Durand et al., Nucleic Acids Res. 1990,18:6353; McCurdy et al., Nucleosides &
Nucleotides 1991,10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et
al.,
'0 Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO
89/02439;
Usman et al., International Publication No. WO 95/06731; Dudycz et al.,
International
Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991,
113:4000, all hereby incorporated by reference herein. Non-nucleotide linkers
can be any
molecule, which is not an oligomeric sequence, that can be covalently coupled
to an
5 oligomeric sequence. Preferred non-nucleotide linkers are oligomeric
molecules formed of
non-nucleotide subunits. Examples of such non-nucleotide linkers are described
by
Letsinger and Wu, (J. Am. Chem. Soc. 117:7323-7328 (1995)), Benseler et al.,
(J. Anu.
Chem. Soc. 115:8483-8484 (1993)) and Fu et al. , (J. Am. Chem. Soc. 116:4591-
4598
(1994)). Preferred non-nucleotide linkers, or subunits for non-nucleotide
linkers, include
0 substituted or unsubstituted C1-Clo straight chain or branched alkyl,
substituted or
unsubstituted CZ-Clo straight chain or branched alkenyl, substituted or
unsubstituted Cz-
Clo straight chain or branched alkynyl, substituted or unsubstituted C1-Clo
straight chain or
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118
branched alkoxy, substituted or unsubstituted CZ-Clo straight chain or
branched
alkenyloxy, and substituted or unsubstituted Cz-Clo straight chain or branched
alkynyloxy.
The substituents for these preferred non-nucleotide linkers (or subunits) can
be halogen,
cyano, amino, carboxy, ester, ether, carboxamide, hydroxy, or mercapto. Thus,
in a
preferred embodiment, the invention features an enzymatic nucleic acid
molecule having
one or more non-nucleotide moieties, and having enzymatic activity to cleave
an RNA or
DNA molecule. By the term "non-nucleotide" is meant any group or compound
which can
be incorporated into a nucleic acid chain in the place of one or more
nucleotide units,
including either sugar and/or phosphate substitutions, and allows the
remaining bases to
0 exhibit their enzymatic activity. The group or compound is abasic in that it
does not
contain a commonly recognized nucleotide base, such as adenosine, guanine,
cytosine,
uracil or thymine. The terms "abasic" or "abasic nucleotide" as used herein
encompass
sugar moieties lacking a base or having other chemical groups in place of
nucleotide base
at the 1' position.
5 In a preferred embodiment, the invention features modified ribozymes with
phosphate backbone modifications comprising one or more phosphorothioate,
phosphorodithioate, methylphosphonate, morpholino, amidate carbamate,
carboxymethyl,
acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,
thioformacetal,
and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone
modifications
'.0 see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and
Properties, in
Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel
Backbone
Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense
Research,
ACS, 24-39.
In a further preferred embodiment of the instant invention, an inverted deoxy
abasic
.5 moiety is utilized at the 3' end of the enzymatic nucleic acid molecule.
By "pyrimidines" is meant nucleotides comprising modified or unmodified
derivatives of a six membered pyrimidine ring. An example of a pyrimidine is
modified or
unmodified uridine.
In a preferred embodiment, the nucleosides of the instant invention include,
2'-O-
methyl-2,6-diaminopurine riboside; 2'-deoxy-2'amino-2,6-diaminopurine
riboside; 2'-(N
alanyl) amino-2'-deoxy-uridine; 2'-(N phenylalanyl)amino-2'-deoxy-uridine; 2'-
deoxy -2'-
(N beta-alanyl) amino ; 2'-deoxy-2'-(lysiyl) amino uridine; 2'-C-allyl
uridine; 2'-O-amino-
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uridine; 2'-O-methylthiomethyl adenosine; 2'-O-methylthiomethyl cytidine ; 2'-
O-
methylthiomethyl guanosine; 2'-O-methylthiomethyl-uridine; 2'-Deoxy-2'-(N
histidyl)
amino uridine; 2'-deoxy-2'-amino-5-methyl cytidine; 2'-(N ~i-carboxamidine-
beta-
alanyl)amino-2'-deoxy-uridine; 2'-deoxy-2'-(N-beta-alanyl)-guanosine; 2'-O-
amino-
adenosine; 2'-(N lysyl)amino -2'-deoxy-cytidine; 2'-Deoxy -2'-(L-histidine)
amino
Cytidine; and 5-Imidazoleacetic acid 2'-deoxy-5'-triphosphate uridine.
By "oligonucleotide" as used herein is meant a molecule having two or more
nucleotides. The polynucleotide can be single, double or multiple stranded and
may have
modified or unmodified nucleotides or non-nucleotides or various mixtures and
0 combinations thereof.
In a preferred embodiment, the enzymatic nucleic acid molecule of formula 1 or
2
include at least three ribonucleotide residues, preferably 4, 5, 6, 7, 8, 9,
and 10
ribonucleotide residues.
In preferred embodiments, the enzymatic nucleic acid of the instant invention
5 includes one or more stretches of RNA, which provide the enzymatic activity
of the
molecule, linked to the non-nucleotide moiety. The necessary RNA components
are
known in the art (see for e.g., Usman et al., supra).
Thus, in one preferred embodiment, the invention features enzymatic nucleic
acid
molecules that inhibit gene expression and/or cell proliferation in vitro or
in vivo (e.g. in
0 patients). These chemically or enzymatically synthesized nucleic acid
molecules contain
substrate binding domains that bind to accessible regions of specific target
nucleic acid
molecules. The nucleic acid molecules also contain domains that catalyze the
cleavage of
target. Upon binding, the enzymatic nucleic acid molecules cleave the target
molecules,
preventing for example, translation and protein accumulation. In the absence
of the
5 expression of the target gene, cell proliferation, for example, is
inhibited.
In another preferred embodiment, catalytic activity of the molecules described
in the
instant invention can be optimized as described by Draper et al., supra. The
details will
not be repeated here, but include altering the length of the ribozyme binding
arms, or
chemically synthesizing ribozymes with modifications (base, sugar and/or
phosphate) that
0 prevent their degradation by serum ribonucleases and/or enhance their
enzymatic activity
(see e.g., Eckstein et al., International Publication No. WO 92/07065;
Perrault et al., 1990
Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren,
1992
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Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No.
WO 93/15187; and Rossi et al., International Publication No. WO 91/03162;
Sproat, US
Patent No. 5,334,711; and Burgin et al., supra; all of these describe various
chemical
modifications that can be made to the base, phosphate and/or sugar moieties of
enzymatic
RNA molecules). Modifications which enhance their efficacy in cells, and
removal of
bases from stem loop structures to shorten RNA synthesis times and reduce
chemical
requirements are desired. (All these publications are hereby incorporated by
reference
herein.).
By "nucleic acid catalyst" as used herein is meant a nucleic acid molecule
(e.g., the
molecule of formulae 1 and 2) capable of catalyzing (altering the velocity
and/or rate of) a
variety of reactions including the ability to repeatedly cleave other separate
nucleic acid
molecules (endonuclease activity) in a nucleotide base sequence-specific
manner. Such a
molecule with endonuclease activity may have complementarity in a substrate
binding
region to a specified gene target, and also has an enzymatic activity that
specifically
cleaves RNA or DNA in that target. That is, the nucleic acid molecule with
endonuclease
activity is able to intramolecularly or intermolecularly cleave RNA or DNA and
thereby
inactivate a target RNA or DNA molecule. This complementarity functions to
allow
sufficient hybridization of the enzymatic RNA molecule to the target RNA or
DNA to
allow the cleavage to occur. 100% complementarity is preferred, but
complementarity as
low as 50-75% may also be useful in this invention. The nucleic acids may be
modified at
the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid as
used herein
is used interchangeably with phrases such as ribozymes, catalytic RNA,
enzymatic RNA,
catalytic oligonucleotides, nucleozyme, RNA enzyme, endoribonuclease,
endonuclease,
minizyme, oligozyme, finderon or nucleic acid catalyst. All of these
terminologies
describe nucleic acid molecules of the instant invention with enzymatic
activity. The
specific examples of enzymatic nucleic acid molecules described in the instant
application
are not limiting in the invention and those skilled in the art will recognize
that all that is
important in an enzymatic nucleic acid molecule of this invention is that it
has a specific
substrate binding site which is complementary to one or more of the target
nucleic acid
regions, and that it have nucleotide sequences within or surrounding that
substrate binding
site which impart a nucleic acid cleaving activity to the molecule (Cech et
al., U.S. Patent
No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).
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The enzymatic nucleic acid molecule of Formula 1 or 2 may independently
comprise
a cap structure which may independently be present or absent.
By "chimeric nucleic acid molecule" or "mixed polymer" is meant that, the
molecule
may be comprised of both modified or unmodified nucleotides.
In yet another preferred embodiment, the 3'-cap is selected from a group
comprising,
4',5'-methylene nucleotide; I-(beta-D-erythrofuranosyl) nucleotide; 4'-thio
nucleotide,
carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl
phosphate, 3-
aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate;
hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-
nucleotide;
modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide;
acyclic
3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl
nucleotide, S'-5'-
inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate;
5'-
phosphorothioate; 1,4-butanediol phosphate; S'-amino; bridging and/or non-
bridging 5'-
phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non
bridging
methylphosphonate and 5'-mercapto moieties (for more details, see Beaucage and
Iyer,
1993, Tetrahedron 49, 1925; incorporated by reference herein). By the term
"non-
nucleotide" is meant any group or compound which can be incorporated into a
nucleic acid
chain in the place of one or more nucleotide units, including either sugar
and/or phosphate
substitutions, and allows the remaining bases to exhibit their enzymatic
activity. The
group or compound is abasic in that it does not contain a commonly recognized
nucleotide
base, such as adenosine, guanine, cytosine, uracil or thymine. The terms
"abasic" or
"abasic nucleotide" as used herein encompass sugar moieties lacking a base or
having
other chemical groups in place of a base at the 1' position.
In a preferred embodiment, the invention features 1-(beta-D-xylofuranosyl)-
xypoxanthine phosphoramidite and a process for the synthesis thereof and
incorporation
into oligonucleotides, such as enzymatic nucleic acid molecule.
In yet another preferred embodiment, the invention features enzymatic nucleic
acid
molecules targeted against HERZ RNA, specifically, ribozymes in the hammerhead
and
NCH motifs.
In a preferred embodiment, the invention features enzymatic nucleic acid
molecules
targeted against PKC alpha RNA, specifically, ribozymes in the hammerhead and
NCH
motifs.
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Targets, for example PKC alpha RNA, for useful ribozymes and antisense nucleic
acids can be determined, for example, as described in Draper et al., WO
95/04818;
McSwiggen et al., U.S. Patent Nos.. 5,525,468 and 5,646,042, all are hereby
incorporated
by reference herein in their totality. Other examples include the following
PCT
applications, which concern inactivation of expression of disease-related
genes: WO
95/23225, WO 95/13380, WO 94/02595, all incorporated by reference herein.
The specific enzymatic nucleic acid molecules described in the instant
application
are not limiting in the invention and those skilled in the art will recognize
that all that is
important in an enzymatic nucleic acid molecule of this invention is that it
has a specific
0 substrate binding site (e.g., D and E of Formula 1 above) which is
complementary to one
or more of the target nucleic acid regions, and that it have nucleotide
sequences within or
surrounding that substrate binding site which impart a nucleic acid cleaving
activity to the
molecule.
All naturally occurring hammerhead ribozymes have an A'SV-Ui6.i base pair. In
5 addition, it is known that substrates for ribozymes based on the consensus
hammerhead
sequence strongly prefer a substrate that contains an N'6.zUi6.iHm triplet in
which H" is
not a guanosine (Koizumi et al., FEBS Lett. 228, 228-230 (1988); Ruffner et
al.,
Biochemistry 29, 10695-10702 (1990); Pernman et al., Gene 113, 157-163
(1992)). Many
experiments have been done in an attempt to isolate ribozymes which are able
to
.0 efficiently relieve the requirement of a U at position 16.1, however,
attempts to fmd
ribozymes which can cleave substrates having a base other than a U at position
16.1 have
proven largely unsuccessful (Perriman et al., Gene 113, 157-163 1992, Singh et
al.,
Antisense and Nucleic Acid Drug Development 6:165-168 (1996)).
However, examination of the recently published X-ray crystal structures (Pley
et al.,
5 Nature 372:68-74 (1994), Scott et al., Cell 81:991-1002 (1995), and Scott et
al., Science
274:2065-2069 (1996)) led to the realization that the A'S.'-U16.1 interaction
is a non
standard base pair with a single hydrogen bond between the exocyclic amine
(N6) of the
adenosine and the 4-oxo group of the uridine. Modeling studies (based on the
crystal
structure) then led to the discovery that the interaction of the wild-type
A'S.i-Ui6.i base pair
0 can be spatially mimicked by replacement with an I'S''-C'6.' base pair that
adopts an
isostructural orientation and which preserves the required contact of the 2-
keto group of
C'6'' with A6 of the uridine turn. In the model, the polarity of the
stabilizing hydrogen
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bond between positions 15.1 and 16.1 is reversed in the hs.i-Cib.i
interaction, but the
correct orientation of the bases around this bond is maintained.
It has been discovered that hammerhead ribozyme analogues containing an
inosine at
position 15.1 readily cleave RNA substrates containing an N'6.zC16.iH1~
triplet. Based on
this, disclosed are compositions, preferably synthetic oligomers, which cleave
a nucleic
acid target sequence containing the triplet N16.2Ci6.iHp It is preferred that
Hl' is not
guanosine, however, under certain circumstances, NCG triplet containing RNA
can be
cleaved by the ribozymes of the instant invention. The ability to cleave
substrates having
N16.2C16.1x17 mplets effectively doubles the number of targets available for
cleavage by
0 compositions of the type disclosed.
Example 10: Synthesis of 1-(beta-D-xylofuranosyl)-xypoxanthine phosphoramidite
Referring to Figure 9, Inosine (1) was 5'-O-monomethoxytritylated and 2'-O-
silylated under standard conditions to afford 2 (Charubala, R; Pfleiderer, W.
Heterocycles
5 1990, 30, 1141). Oxidation/reduction procedure afforded 3 in moderate yield
(Matulic-
Adamic, J.; Daniher, A.T.; Gonzalez, C.; Beigelman, L. Bioorg. Med. Chem.
Lett.. 1999,
9, 157): 1H NMR (CDCl3) 8 12.80 (br s, 1H, NH), 8.11 (s, 1H, H-8), 8.08 (s,
1H, H-2),
7.45-6.80 (m, 14H, trityl), 5.85 (d, J~.~2~= 1.6, 1H, H-1'), 4.83 (d,
J2y3~=7.2, 1H, H-2'),
4.46 (br s, 1H, 3'-OH), 4.34 (m, 1H, H-4'), 4.06 (m, 1H, H-3'), 3.77 (s, 6H, 2
x OMe),
0 3.60 (app d, 2H, H-5', H-5"), 0.89 (s, 9H, t-Bu), 0.07 (s, 3H, Me), 0.06 (s,
3H, Me).
Standard phosphitylation of 3 afforded the desired phosphoramidite 4.
More acid stable 5'-O-MMT group is used in this particular case because
applicant
found that 5'-O-DMT protection is more labile in xylo nucleoside series than
in ribo
nucleoside series.
5 The xylo-inosine was incorporated into oligonucleotides using the standard
procedures known in the art and as described herein.
Example 11: Activity of the xylo-Inosine-modified NCH Ribozyme
Several NCH ribozymes with xylo-inosine at position 15.1 were designed (Figure
7)
0 to cleave RNA containing GCA, ACA, UCA or the CCA triplet. These ribozymes
were
CA 02403243 2002-02-21
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124
synthesized and purified as described herein and tested using standard RNA
cleavage
reaction conditions (see Table 31, for example, and see below).
The ribozymes were chemically synthesized. The method of synthesis used
followed
the procedure for normal RNA synthesis as described above and in Usman et al.,
( 1987 J.
Am. Chem. Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18,
5433) and
Wincott et al., supra, and made use of common nucleic acid protecting and
coupling
groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-
end. The
average stepwise coupling yields were >98%.
Ribozymes were purified by gel electrophoresis using general methods or were
I 0 purified by high pressure liquid chromatography (HPLC; See Wincott et al.,
supra; the
totality of which is hereby incorporated herein by reference) and were
resuspended in
water. The sequences of the chemically synthesized ribozymes used in this
study are
shown below in Table 33.
Cleavage Reactions: Full-length or partially full-length, internally-labeled
target
5 RNA for ribozyme cleavage assay is prepared by in vitro transcription in the
presence of
[alpha-32p] CTP, passed over a G 50 Sephadex column by spin chromatography and
used
as substrate RNA without further purification. Alternately, substrates were 5'-
32P-end
labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-
warming a
2X concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-
HCI, pH
!0 7.5 at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by
adding the 2X
ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was
also
pre-warmed in cleavage buffer. As an initial screen, assays are carried out
for 1 hour at
37°C using a final concentration of 40 nM or 1 mM ribozyine, i.e.,
ribozyme excess. The
reaction is quenched by the addition of an equal volume of 95% formamide, 20
mM
!5 EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample
is
heated to 95°C for 2 minutes, quick chilled and loaded onto a
denaturing polyacrylamide
gel. Substrate RNA and the specific RNA cleavage products generated by
ribozyme
cleavage are visualized on an autoradiograph of the gel. The percentage of
cleavage is
determined by Phosphor Imager~ quantitation of bands representing the intact
substrate
.0 and the cleavage products.
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The results of the experiments are summarized in Table 32, which shows that
NCH-
xylo ribozymes are catalytically active to cleave target RNA.
Example 12: Activity of NCH Ribozyme variants
The nucleic acid molecules of the instant invention allow for the ability to
cleave a
new set of 12 NCH triplets. Determination of single turnover rate constants at
pH 6 of
these ribozymes in the all ribo form show that with NCA type triplets, the
cleavage rate is
higher than at NUA sites. NCC and NUC site rates are similar, and NCU sites
are slightly
lower than NUU sites. Additional measurements of multiple turnover parameters
of the all
0 ribo ribozymes performed under non-saturating conditions using SnM ribozyme
and
changing the substrate concentration from 50 to 500 nM at pH 7.4 with 10 mM Mg
++ at
37 °C gave Km= 100 nM and kcat=6.5 min -~ for GCA vs Km =30 nM and kcat
=2.0 min
-1 for GUA cleaving all ribo ribozymes. These data verify that the ribozymes
with an I~C
base pair are efficient catalysts in multiple turnover reactions and the
relative order of
5 activity between NCH and NUH cleavers established at pH 6 (Ludwig et al.,
1998, Nucleic
Acids Res., 26, 2279-2285) remains unchanged.
To gain more insight into the structural requirements of the 15.1- 16.1 base
pair of
the ribozymes of the instant invention, applicant synthesized several variants
of the active
I-15.1 ~C-16.1 structure and tested these ribozyme analogues with their
corresponding
.0 substrates. The influence of several core stabilization strategies on the
activity of the NCH
cleaving ribozymes was also investigated.
Various nucleoside analogs were incorporated at position 1 S.1 of the
ribozyme.
Cleavage activity was tested with the complementary Fl* labeled substrates at
pH 7.4 in
the presence of 10 mM Mg ~ under conditions of ribozyme excess (i.e. single
turnover
5 conditions). The modified oligonucleotides were synthesized by standard
oligonucleotide
synthesis procedures. Xanthosine was protected using O-2 ,O-4
pivaloyloxymethyl groups;
N,N-dimethylguanosine with 6-O-( 2-nitrophenyl-)ethyl and 6-thio-inosine with
S-
cyanoethyl protecting groups. The cleavage activity of the ribozymes
containing the 15.1
analogs is summarized in Figure 36. For comparison Figure 37 summarizes
reported
0 functional group modification studies performed at the A 15.1 residue in the
A-15.1 ~U-
16.1 context of NUH cleaving ribozymes.
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Modifications at the purine 15.1 N1 and/or C6 positions (Figure 36 A, B, C)
In the 6-thio-inosine (A) (s1) 15.1 substituted ribozyme, the original (I-
15.1) position
6 O~H-N (C-16.1) bonds are replaced by weaker (sI-15.1) position 6 S~H-N (C-
16.1)
hydrogen bonds while all other functional groups remain unchanged. Ribozymes
with an
adenosine (B) at position 15.1 (A-15.1) are inactive with C-16.1 substrates
since the
ribozyme geometry requires the [A-1 S.1 ] position 6 amino group and the [C-
16.1 ] position
4 amino group hydrogen-bond donor functional groups to be in close proximity.
Similarly,
low activity is observed with I-15.1 ribozymes and U-16.1 substrates, where
the [I-15.1]
position 6 keto and [C-16.1] position 4 keto hydrogen-bond acceptor groups are
opposed
(Figure 37, B). Although inosine can form stable mismatch pairs with uridine
in RNA
duplexes or in tRNA anticodon-mRNA interactions, these results suggest that
the
geometry in the I~U mismatches differ from that of the A~U (or I~C) base pair
in the active
NUH ribozyme. Substitution of N1-Methyl-inosine (C) in place of inosine at
position 15.1
leads to complete loss of cleavage activity.
Modifications at the purine 1 S.1 C2 and/or N3 position (Figure 36 D, E, F)
The extremely low activity observed with the G-15.1 (D) substituted analog may
be
explained by the formation of a G-C Watson-Crick base pair. The replacement of
the I~C
pair with a G~C pair can significantly distort the geometry at the 15.1-16.1
position. G-
15.1 N2-alkylation (E) gives only minimal recovery of catalytic activity
compared to G-
15. l, suggesting that the steric problems introduced by the bulky N-methyl
groups may
interfere with stacking interactions. The activity of this construct is
significantly less than
that of iso-G-15.1 (Figure 37, E) containing ribozymes in the standard A-U
context.
Xanthosine 15.1 (F) contains the same functional groups as inosine at the N1
and C6 sites
but contains an additional hydrogen-bond donor site at position N3 along with
a C2
carbonyl group. The complete lack of activity seen with this construct
reinforces the
importance of the purine N3 acceptor functionality in transition state
formation. Similarly,
3-deaza-adenosine (Figure 37, F) containing ribozymes were also inactive. The
C2
carbonyl of the 15.1 purine shows no significant negative interference in iso-
guanosine
containing 15.1 ribozymes.
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Activity of modified core variants
To complete the characterization of the I~C pair containing ribozymes, the
acceptance of various core substitution patterns was tested. Short substrates
containing
GCH and GUH (H= non G) triplets were compared using 3 different modified
ribozymes.
The acceptance of the U-4 2'-O-alkyl substituent is the greatest with GCA
triplets while U-
4= 2'-deoxy-2'-amino uridine and U-4= ribo uridine substituted ribozymes show
a similar
level of activity with NCH and NUH triplets. The results of this comparison
are
summarized in Table 64. In addition, a ribozyme construct in which ribo
inosine replaces
adenosine at positions 14 and 15.1 was tested which demonstrated cleavage
activity.
Apart from the A-15.1 ~U-16.1 to I-15 .1 ~C-16.1 change that reverses the
polarity
of an important H-bond in the ribozyme structure, no other functional group
changes at
the 15.1 purine residue seem to be compatible with the requirements of
efficient
catalysis. The I-15.1 and A-15.1 ribozymes are equally suitable for practical
applications because there are only minor differences in the acceptance of
stabilizing
residues.
Example 13 : Activity of NCH Ribozyme to inhibit HERZ eg ne expression
Applicant has designed, synthesized and tested several NCH ribozymes and HH
ribozymes targeted against HER2 RNA (see, for example, Tables 31 and 34) in
cell
proliferation assays.
Proliferation assay: The model proliferation assay used in the study can
require a
cell plating density of 2000 cells/well in 96-well plates and at least 2 cell
doublings over a
S-day treatment period. To calculate cell density for proliferation assays,
the FIPS (fluoro-
imaging processing system) method well in the art was used. This method allows
for cell
density measurements after nucleic acids are stained with CyQuant~ dye, and
has the
advantage of accurately measuring cell densities over a very wide range 1,000-
100,000
cells/well in 96-well format.
Ribozymes (50-200 nM) were delivered in the presence of cationic lipid at 2.0
~g/mL and inhibition of proliferation was determined on day 5 post-treatment.
Two full
ribozyme screens were completed and 4 lead HH and 11 lead NCH ribozymes were
chosen for further testing. Of the 15 lead Rzs chosen from primary screens, 4
NCH and 1
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HH Rzs continued to inhibit cell proliferation in subsequent experiments. NCH
Rzs
against sites, 2001 (RPI No. 17236), 2783 (RPI No. 17249), 2939 (RPI No.
17251) or
3998 (RPI No. 17262) caused inhibition of proliferation ranging from 25-60% as
compared to a scrambled control Rz (IA; RPI No. 17263). Of the five lead Rzs,
the most
efficacious is the NCH Rz (RPI No. 17251) against site 2939 of HER2 RNA. An
example of results from cell culture assay is shown in Figure 3. Referring to
Figure 3,
NCH ribozymes and a HH ribozyme targeted against HER2 RNA are shown to cause
significant inhibition of proliferation of cells. This shows that ribozymes,
for instance, the
NCH ribozymes are capable of inhibiting HER2 gene expression in mammalian
cells.
0
Example 14: Activity of NCH Ribozyme to inhibit PKC alpha ene expression
The Protein Kinase C family contains twelve currently known isozymes divided
into
three classes: the classic, Cap dependent (PKCa, ~iI, ~3II, y), the novel, non-
Cap
dependent (PKCB, E, p, r), 0) and the atypical (PKC ~, i/~,); all of which are
5 serine/threonine kinases. These isozymes show distinct and overlapping
tissue, cellular,
and subcellular distribution. They aid in the regulation of cell growth and
differentiation
through their response to second messenger products of lipid metabolism
(Blobe, et al.,
1996, Cancer Surveys, 27, 213-248). These second messengers include
diacylglyceral
(DAG), inositol-triphosphate (IP3), lysophospholipids, free fatty acids, and
phosphatidate
'.0 which act directly or in addition to changes in the Cap concentration. A
simple model for
PKCa activation follows a two step mechanism. First, membrane association of
PKCa is
through Cap and phospholipid interactions and second, the kinase is activated
by
interaction with DAG. An example of a signal cascade subsequent to PKC
activation is
PKC's phosphorylation of c-Raf, which phosphorylates MEK, which phosphorylates
'5 MAP, which phosphorylates transcription factors such as Jun and thereby
activates a
mitogenic program in the nucleus. There are numerous substrates for the
various PKC's,
one which for PKCa ultimately stimulates transcription factors that activate P-
glycoprotein (P-gp) causing the multi-drug resistant phenotype (MDR) (Blobe,
et al.,
1994, Cancer and Metastasis Reviews, 13, 411-431).
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Cell Culture Review
PKC's have been implicated in tumor promotion since the discovery that these
molecules can serve as receptors for tumor-promoting phorbol esters. An
increase in PKC
overexpression in numerous tumor cell lines and tumor tissues has also been
demonstrated. PKC overexpression has been shown to be associated with
increased
invasion and metastasis in mouse Lewis lung carcinoma, mouse B16 melanoma (Lee
et al.,
1997, Molecular Carcinogenesis, 18, 44-53), mouse mammary adenocarcinoma,
mouse
fibrosarcoma, human lung carcinoma (Wang and Liu, 1998, Acta Pharmacologica
Sinica,
19, 265-268), human bladder carcinoma, human pancreatic cancer (Denham et al.,
1998,
0 Surgery, 124, 218-223), and human gastric cancer (Dean et al., 1996, Cancer
Research,
56, 3499-3507). Mounting evidence suggests PKCa can stimulate adhesion
molecule
expression and can directly act on these membrane bound species as substrates,
thereby
modulating cellular adhesion to the extracellular matrix and increasing
metastic potential.
Furthermore, human surgical specimens have demonstrated elevated PKC in breast
5 tumors, thyroid carcinomas and melanomas (Becker et al., 1990, Oncogene, 5,
1133-
1139).
Utz et al., 1994, Int. J. Cancer, 57, 104-110, describe a cell proliferation
assay in
which small molecule inhibitors of PKC demonstrate anti-proliferative activity
in CCRF-
VCR 1000 and KB-8511 cells with the multidrug resistant (MDR) phenotype. PKCa
is
.0 overexpressed in tumor tissues that express the MDR phenotype. This
phenotype is
associated with the expression of a 170 kDa broad specificity drug efflux
pump, P-gp.
PKCa phosphorylation of P-gp has been shown in vitro. In addition, PKC
expression
correlates with resistance to doxorubicin and high P-gp levels in human renal
carcinoma
and non-small cell lung carcinoma. Inhibitors of PKC partially reverse the MDR
5 phenotype and decrease phosphorylation of P-gp (Caponigro et al., 1997, Anti-
Cancer
Drugs, 8, 26-33).
Dean et al., 1994, Journal of Biological Chemistry, 269, 16416-24, describe
cell
culture studies in which antisense targeting of PKC a resulted in the potent
inhibition of
mRNA and protein expression in human lung carcinoma (A549) cells. In this
study, PKC
0 a inhibition resulted in the reduced induction of intercellular adhesion
molecule 1 (ICAM-
1) mRNA by phorbol esters.
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Yano et al., 1999, Endocrinology, 140, 4622-4632, describe a cell
proliferation study
in which down regulation of different PKC isoforms, including PKCa, results in
the
inhibition of insulin like growth factor I induced vascular smooth muscle cell
proliferation,
migration, and gene expression.
Wang et al., 1999, Experimental Cell Research, 250, 253-263, describe cell
culture
studies in which antisense inhibition of PKCa results in the reversal of the
transformed
phenotype in human lung carcinoma (LTEPa-2) cells. In this study, the amounts
of PKCa
protein and total PKC activity were decreased when compared to control cells.
Sioud and Sorensen, 1998, Nature Biotechnology, 16, 556-561, describe
0 hammerhead ribozyme inhibition of PKCa in rat glioma cell lines (BT4C and
BT4Cn).
This study demonstrated inhibition of malignant glioma cell proliferation
along with the
inhibition of regulatory Bcl-xL protein expression. Bcl-xL is overexpressed in
glioma cells
and is an apoptosis inhibitor. The ribozyme mediated inhibition of cell
proliferation
presumably results from apoptosis induction of transformed glioma cells
through
5 suppression of PKCa and Bcl-xL (Leirdal and Sioud, 1999, British J. of
Cancer, 80, 1558-
1564).
Animal Models
Evaluating the efficacy of anti-PKCa agents in animal models is an important
prerequisite to human clinical trials. A variety of mouse xenograft models
using human
:0 tumor cell lines have been developed using cell lines which express high
levels of PKCa
protein. McGraw et al, 1997, Anti-Cancer Drug Design, 12, 315-326, describe
mouse
xenograft models using human breast (MDA MB-321), prostate (Du-145), colon
(Colo
205, WiDr), lung (NCI H69, H209, J460, H520, A549), bladder (T-24), and
melanoma
(SK-mel 1 ) carcinoma cells. Antisense oligonucleotides targeting PKCa
administered
5 intravenously following s.c. transplanted tumor cells resulted in dose
dependant decreases
in tumor size when compared to controls in most cases. Similar studies using T-
24
bladder carcinoma, non-small cell lung carcinoma (A549), and Colo 205 colon
carcinoma
mouse xenografts are described in Dean et al, 1996, Biochemical Society
Transactions, 24,
623. Sioud and Sorensen, 1998, Nature Biotechnology, 16, 556-561, describe a
rat model
in which inbred syngeneic BDIX rats were inoculated subcutaneously with BT4Cn
glioma
cells. After approximately three weeks, rats were treated with a single
injection of
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ribozyme targeting PKCa resulting in inhibition of tumor growth as determined
by tumor
size and/or weight when compared to controls. The above studies provide proof
that
inhibition of PKCa expression by anti-PKCa agents causes inhibition of tumor
growth in
animals. Lead anti-PKCa ribozymes chosen from in vitro assays can be further
tested in
mouse xenograft models. Ribozymes can be first tested alone and then in
combination
with standard chemotherapies.
Animal Model Development
Human lung (A549, NCI H520) tumor and breast (MDA-MB 231) cell lines can be
characterized to establish their growth curves in mice. These cell lines are
been implanted
into both nude and SC1D mice and primary tumor volumes are measured 3 times
per week.
Growth characteristics of these tumor lines using a Matrigel implantation
format can also
be established. In addition, the use of other cell lines that have been
engineered to express
high levels of PKCa can also be used. The tumor cell lines) and implantation
method
that supports the most consistent and reliable tumor growth can be used in
animal studies
to test promising PKCa ribozyme(s). Ribozymes can be administered by daily
subcutaneous injection or by continuous subcutaneous infusion from Alzet mini
osmotic
pumps beginning 3 days after tumor implantation and continuing for the
duration of the
study. Group sizes of at least 10 animals are employed. Efficacy is determined
by
statistical comparison of tumor volume of ribozyme-treated animals to a
control group of
animals treated with saline alone. Because the growth of these tumors is
generally slow
(45-60 days), an initial endpoint will be the time in days it takes to
establish an easily
measurable primary tumor (i.e. 50-100 mm3) in the presence or absence of
ribozyme
treatment.
Clinical Summary
Overview
Ribozymes targeting PKCa have strong potential to develop into useful
therapeutics
directed towards numerous cancer types. Lung cancer is the leading cause of
cancer deaths
for both men and women in the USA. The incidence of lung cancer in the United
States is
172,000 cases per year, accounting for 14% of cancer diagnoses. Approximately
158,000
die each year of lung cancer, accounting for 28% of all cancer deaths.
Numerous other
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indications exist including cancers of the bladder, colon, breast, prostate,
and ovary in
addition to melanoma and glioblastoma.
McGraw et al., 1997, Anti-Cancer Drug Design, 12, 315-326, describe a Phase I
trial
for ISIS 3521/CGP 64128A, a PKC alpha antisense construct. In this trial, ISIS
3521/CGP
64128A was administered as either a two-hour i.v. infusion three times per
week for three
consecutive weeks, or as a continuous i.v. infusion for twenty-one consecutive
days. The
authors report that patients demonstrated excellent tolerance to the antisense
compound
when administered at doses of up to 2.5 mg/kg by the two-hour i.v. infusion
and at 1.5
mg/kg/day by continuous i.v. infusion. In patients receiving the two-hour i.v.
infusion
schedule, the post-infusion plasma concentration of the compound increased
proportional
to the dose, and metabolites were determined to have been cleared rapidly from
plasma
with a half life of thirty to forty-five minutes. These metabolites were
composed of chain-
shortened oligonucleotides, consistent with exonuclease-mediated degradation.
No
evidence of accumulation, induction, or inhibition of metabolism was found
after the
administration of repetitive doses.
Therapy
Treatment options for lung cancer are determined by the type and stage of the
cancer
and include surgery, radiation therapy, and chemotherapy. For many localized
cancers,
surgery is usually the treatment of choice. Because the disease has usually
spread by the
time it is discovered, radiation therapy and chemotherapy are often needed in
combination
with surgery. Chemotherapy alone or combined with radiation has replaced
surgery as the
treatment of choice for small cell lung cancer; on this regimen, a large
percentage of
patients experience remission, which in some cases is long-lasting. The 1-year
relative
survival rates for lung cancer have increased from 32% in 1973 to 41 % in
1994, largely
due to improvements in surgical techniques. The 5-year relative survival rate
for all stages
combined is only 14%. The survival rate is 50% for cases detected when the
disease is still
localized, but only 15% of lung cancers are discovered that early.
Common chemotherapies include various combinations of cytotoxic drugs to kill
the
cancer cells. These drugs include paclitaxel (Taxol), docetaxel, cisplatin,
methotrexate,
cyclophosphamide, doxorubin, fluorouracil etc. Significant toxicities are
associated with
these cytotoxic therapies. Well-characterized toxicities include nausea and
vomiting,
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myelosuppression, alopecia and mucosity. Serious cardiac problems are also
associated
with certain of the combinations, e.g. doxorubin and paclitaxel, but are less
common.
Applicant has designed several NCH ribozymes targeted against PKCa RNA
(Genebank accession No NM_002737) (see, for example, Table 63). These
ribozymes are
used first in a proliferation assay that is used to select ribozyme leads.
Proliferation assay: The model proliferation assay useful in the study can
require a
cell plating density of 2000 cells/well in 96-well plates and at least 2 cell
doublings over a
5-day treatment period. To calculate cell density for proliferation assays,
the FIPS (fluoro-
imaging processing system) method well known in the art can be used. This
method allows
I 0 for cell density measurements after nucleic acids are stained with
CyQuant~ dye, and has
the advantage of accurately measuring cell densities over a very wide range
1,000-100,000
cells/well in 96-well format.
Ribozymes (50-200 nM) are delivered in the presence of cationic lipid at 2.0
pg/mL
and inhibition of proliferation is determined on day 5 post-treatment. Two
full ribozyme
I 5 screens are usually completed and lead ribozymes are chosen for further
testing. Of the
lead ribozymes chosen from primary screens, ribozymes which continue to
inhibit cell
proliferation in subsequent experiments are selected for PKCa RNA and protein
inhibition
studies.
!0 Example 15: Nucleoside Triphosphates and their incorporation into
olig_onucleotides
The synthesis of nucleotide triphosphates and their incorporation into nucleic
acids
using polymerise enzymes has greatly assisted in the advancement of nucleic
acid
research. The polymerise enzyme utilizes nucleotide triphosphates as precursor
molecules
to assemble oligonucleotides. Each nucleotide is attached by a phosphodiester
bond
!5 formed through nucleophilic attack by the 3' hydroxyl group of the
oligonucleotide's last
nucleotide onto the 5' triphosphate of the next nucleotide. Nucleotides are
incorporated
one at a time into the oligonucleotide in a 5' to 3' direction. This process
allows RNA to
be produced and amplified from virtually any DNA or RNA templates.
Most natural polymerise enzymes incorporate standard nucleotide triphosphates
into
.0 nucleic acid. For example, a DNA polymerise incorporates dATP, dTTP, dCTP,
and
dGTP into DNA and an RNA polymerise generally incorporates ATP, CTP, UTP, and
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GTP into RNA. There are however, certain polymerises that are capable of
incorporating
non-standard nucleotide triphosphates into nucleic acids (Joyce, 1997, PNAS
94, 1619-
1622, Huang et al., Biochemistry 36, 8231-8242).
Before nucleosides can be incorporated into RNA transcripts using polymerise
enzymes they must first be converted into nucleotide triphosphates which can
be
recognized by these enzymes. Phosphorylation of unblocked nucleosides by
treatment
with POC13 and trialkyl phosphates was shown to yield nucleoside 5'-
phosphorodichloridates (Yoshikawa et al., 1969, Bull. Chem. Soc. (Japan) 42,
3505).
Adenosine or 2'-deoxyadenosine S'-triphosphate was synthesized by adding an
additional
step consisting of treatment with excess tri-n-butylammonium pyrophosphate in
DMF
followed by hydrolysis (Ludwig, 1981, Acta Biochim. et Biophys. Acid. Sci.
Hung. 16,
131-133).
Non-standard nucleotide triphosphates are not readily incorporated into RNA
transcripts by traditional RNA polymerises. Mutations have been introduced
into RNA
polymerise to facilitate incorporation of deoxyribonucleotides into RNA (Sousa
& Padilla,
1995, EMBO J. 14,4609-4621, Bonner et al., 1992, EMBO J. 11, 3767-3775, Bonner
et al.,
1994, J. Biol. Chem. 42, 25120-25128, Aurup et al., 1992, Biochemistry 31,
9636-9641).
McGee et al., International PCT Publication No. WO 95/35102, describes the
incorporation of 2'-NHZ-NTP's, 2'-F-NTP's, and 2'-deoxy-2'-benzyloxyamino UTP
into
RNA using bacteriophage T7 polymerise.
Wieczorek et al., 1994, Bioorganic ~c Medicinal Chemistry Letters 4, 987-994,
describes the incorporation of 7-deaza-adenosine triphosphate into an RNA
transcript
using bacteriophage T7 RNA polymerise.
Lin et al., 1994, Nucleic Acids Research 22, 5229-5234, reports the
incorporation of
2'-NHZ-CTP and 2'-NHz-UTP into RNA using bacteriophage T7 RNA polymerise and
polyethylene glycol containing buffer. The article describes the use of the
polymerise
synthesized RNA for in vitro selection of aptamers to human neutrophil
elastase (HNE).
This invention relates to novel nucleotide triphosphate (NTP) molecules, and
their
incorporation into nucleic acid molecules, including nucleic acid catalysts.
The NTPs of
the instant invention are distinct from other NTPs known in the art. The
invention further
relates to incorporation of these nucleotide triphosphates into
oligonucleotides using an
RNA polymerise; the invention further relates to novel transcription
conditions for the
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incorporation of modified (non-standard) and unmodified NTP's, into nucleic
acid
molecules. Further, the invention relates to methods for synthesis of novel
NTP's
In a first aspect, the invention features NTP's having the formula
triphosphate-OR,
for example the following formula 3:
O O O
-O-P -fl -P -~ -P OR
0- o- o-
where R is any nucleoside; specifically the nucleosides 2'-O-methyl-2,6-
diaminopurine riboside; 2'-deoxy-2'amino-2,6-diaminopurine riboside; 2'-(N
alanyl)
amino-2'-deoxy-uridine; 2'-(N phenylalanyl)amino-2'-deoxy-uridine; 2'-deoxy -
2'-(N [3-
alanyl) amino ; 2'-deoxy-2'-(lysiyl) amino uridine; 2'-C-allyl uridine; 2'-O-
amino-uridine;
0 2'-O-methylthiomethyl adenosine; 2'-O-methylthiomethyl cytidine ; 2'-O-
methylthiomethyl guanosine; 2'-O-methylthiomethyl-uridine; 2'-deoxy-2'-(N
histidyl)
amino uridine; 2'-deoxy-2'-amino-5-methyl cytidine; 2'-(N [i-carboxamidine-[3-
alanyl)amino-2'-deoxy-uridine; 2'-deoxy-2'-(N-(3-alanyl)-guanosine; 2'-O-amino-
adenosine; 2'-(N lysyl)amino-2'-deoxy-cytidine; 2'-Deoxy -2'-(L-histidine)
amino
5 Cytidine; 5-Imidazoleacetic acid 2'-deoxy uridine, 5-[3-(N-4-
imidazoleacetyl)aminopropynyl]-2'-O-methyl uridine, 5-(3-aminopropynyl)-2'-O-
methyl
uridine, 5-(3-aminopropyl)-2'-O-methyl uridine, 5-[3-(N-4-
imidazoleacetyl)aminopropyl]-
2'-O-methyl uridine, 5-(3-aminopropyl)-2'-deoxy-2-fluoro uridine, 2'-Deoxy-2'-
((3-alanyl-
L-histidyl)amino uridine, 2'-deoxy-2'-(3-alaninamido-uridine, 3-(2'-deoxy-2'-
fluoro-[i-D-
ribofuranosyl)piperazino[2,3-D]pyrimidine-2-one, 5-[3-(N-4-
imidazoleacetyl)aminopropyl]-2'-deoxy-2'-fluoro uridine, 5-[3-(N-4-
imidazoleacetyl)aminopropynyl]-2'-deoxy-2'-fluoro uridine, 5-E-(2-carboxyvinyl-
2'-
deoxy-2'-fluoro uridine, 5-[3-(N-4-aspartyl)aminopropynyl-2'-fluoro uridine, S-
(3-
aminopropyl)-2'-deoxy-2-fluoro cytidine, and S-[3-(N-4-succynyl)aminopropyl-2'-
deoxy-
5 2-fluoro cytidine.
In a second aspect, the invention features inorganic and organic salts of the
nucleoside triphosphates of the instant invention.
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In a third aspect, the invention features a process for the synthesis of
pyrimidine
nucleotide triphosphate (such as UTP, 2'-O-MTM-UTP, dUTP and the like)
including the
steps of monophosphorylation where the pyrimidine nucleoside is contacted with
a
mixture having a phosphorylating agent (such as phosphorus oxychloride,
phospho-tris-
triazolides, phospho-tris-triimidazolides and the like), trialkyl phosphate
(such as
triethylphosphate or trimethylphosphate or the like) and a hindered base (such
as
dimethylaminopyridine, DMAP and the like) under conditions suitable for the
formation of
pyrimidine monophosphate; and pyrophosphorylation where the pyrimidine
monophosphate is contacted with a pyrophosphorylating reagent (such as
I 0 tributylammonium pyrophosphate) under conditions suitable for the
formation of
pyrimidine triphosphates.
By "nucleotide triphosphate" or "NTP" is meant a nucleoside bound to three
inorganic phosphate groups at the 5' hydroxyl group of the modified or
unmodified ribose
or deoxyribose sugar where the 1' position of the sugar may comprise a nucleic
acid base
I 5 or hydrogen. The triphosphate portion may be modified to include chemical
moieties
which do not destroy the functionality of the group (i.e., allow incorporation
into an RNA
molecule).
In another preferred embodiment, nucleotide triphosphates (NTPs) of the
instant
invention are incorporated into an oligonucleotide using an RNA polymerase
enzyme.
'.0 RNA polymerases include but are not limited to mutated and wild type
versions of
bacteriophage T7, SP6, or T3 RNA polymerases. Applicant has also found that
the NTPs
of the present invention can be incorporated into oligonucleotides using
certain DNA
polymerases, such as Taq polymerase.
In yet another preferred embodiment, the invention features a process for
'.5 incorporating modified NTP's into an oligonucleotide including the step of
incubating a
mixture having a DNA template, RNA polymerase, NTP, and an enhancer of
modified
NTP incorporation under conditions suitable for the incorporation of the
modified NTP
into the oligonucleotide.
By "enhancer of modified NTP incorporation" is meant a reagent which
facilitates
SO the incorporation of modified nucleotides into a nucleic acid transcript by
an RNA
polymerase. Such reagents include, but are not limited to, methanol, LiCI,
polyethylene
glycol (PEG), diethyl ether, propanol, methyl amine, ethanol, and the like.
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In another preferred embodiment, the modified nucleotide triphosphates can be
incorporated by transcription into a nucleic acid molecules including
enzymatic nucleic
acid, antisense, 2-SA antisense chimera, oligonucleotides, triplex forming
oligonucleotide
(TFO), aptamers and the like (Stun et al., 1995 Pharmaceutical Res. 12, 465).
By "triplex forming oligonucleotides (TFO)" it is meant an oligonucleotide
that can
bind to a double-stranded DNA in a sequence-specific manner to form a triple-
strand
helix. Formation of such triple helix structure has been shown to inhibit
transcription of
the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89,
504).
In yet another preferred embodiment, the modified nucleotide triphosphates of
the
0 instant invention can be used for combinatorial chemistry or in vitro
selection of nucleic
acid molecules with novel function. Modified oligonucleotides can be
enzymatically
synthesized to generate libraries for screening.
In another preferred embodiment, the invention features nucleic acid based
techniques (e.g., enzymatic nucleic acid molecules), antisense nucleic acids,
2-SA
5 antisense chimeras, triplex DNA, antisense nucleic acids containing RNA
cleaving
chemical groups) isolated using the methods described in this invention and
methods for
their use to diagnose, down regulate or inhibit gene expression.
In yet another preferred embodiment, the invention features enzymatic nucleic
acid
molecules targeted against HER2 RNA, specifically including ribozymes in the
class II
'.0 (zinzyme) motif.
Targets, for example HER2 RNA, for useful ribozymes and antisense nucleic
acids
can be determined, for example, as described in Draper et al., WO 93/23569;
Sullivan et
al., WO 93/23057; Thompson et al., WO 94/02595; Draper et al., WO 95/04818;
McSwiggen et al., US Patent Nos. 5,525,468 and 5,646,042, all are hereby
incorporated by
.5 reference herein in their totalities. Other examples include the following
PCT
applications, which concern inactivation of expression of disease-related
genes: WO
95/23225, and WO 95/13380; all of which are incorporated by reference herein.
In yet another preferred embodiment, the invention features a process for
incorporating a plurality of compounds of formula 3.
In yet another embodiment, the invention features a nucleic acid molecule with
catalytic activity having formula 4:
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W
J i
' (Y'Y' )m
i
(X)o
~ Domain A
In the formula shown above X, Y, and Z represent independently a nucleotide or
a
non-nucleotide linker, which may be same or different; ~ indicates hydrogen
bond
formation between two adjacent nucleotides which may or may not be present; Y'
is a
nucleotide complementary to Y; Z' is a nucleotide complementary to Z;1 is an
integer
greater than or equal to 3 and preferably less than 20, more specifically 4,
5, 6, 7, 8, 9, 10,
11, 12, or 15; m is an integer greater than 1 and preferably less than 10,
more specifically
2, 3, 4, 5, 6, or 7; n is an integer greater than 1 and preferably less than
10, more
specifically 3, 4, 5, 6, or 7; o is an integer greater than or equal to 3 and
preferably less
than 20, more specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15;1 and o may
be the same
length (1= o) or different lengths (1 ~ o); each X(1) and X(o) are
oligonucleotides which are
of sufficient length to stably interact independently with a target nucleic
acid sequence (the
target can be an RNA, DNA or RNA/DNA mixed polymers); W is a linker of >_ 2
nucleotides in length or may be a non-nucleotide linker; A, U, C, and G
represent the
nucleotides; G is a nucleotide, preferably 2'-O-methyl or ribo; A is a
nucleotide, preferably
2'-O-methyl or ribo; U is a nucleotide, preferably 2'-amino (e.g., 2'-NHZ or
2'-O- NHz),
2'-O-methyl or ribo; C represents a nucleotide, preferably 2'-amino (e.g., 2'-
NHZ or 2'-O-
(Z'Z' )n
II
W
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NHZ), and represents a chemical linkage (e.g. a phosphate ester linkage, amide
linkage, phosphorothioate, phosphorodithioate or others known in the art).
In yet another embodiment, the invention features a nucleic acid molecule with
catalytic activity having formula 5:
(X)o
W ' ~Z'1 G
Z %~ C
G C U (X)~
A U
G UG
U G
\ Y A i
In the formula shown above X, Y, and Z represent independently a nucleotide or
a
non-nucleotide linker, which may be same or different; ~ indicates hydrogen
bond
formation between two adjacent nucleotides which may or may not be present; Z'
is a
nucleotide complementary to Z;1 is an integer greater than or equal to 3 and
preferably
less than 20, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15; n is an
integer greater than
1 and preferably less than 10, more specifically 3, 4, 5, 6, or 7; o is an
integer greater than
or equal to 3 and preferably less than 20, more specifically 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, or
15;1 and o may be the same length (1= o) or different lengths (1 ~ o); each
X(1~ and X(ot
are oligonucleotides which are of sufficient length to stably interact
independently with a
target nucleic acid sequence (the target can be an RNA, DNA or RNA/DNA mixed
polymers); X~o~ preferably has a G at the 3'-end, X(t~ preferably has a G at
the 5'-end; W
is a linker of >_ 2 nucleotides in length or may be a non-nucleotide linker; Y
is a linker of >_
1 nucleotides in length, preferably G, 5'-CA-3', or 5'-CAA-3', or may be a non-
nucleotide
linker; A, U, C, and G represent nucleotides; G is a nucleotide, preferably 2'-
O-methyl,
2'-deozy-2'-fluoro, or 2'-OH; A is a nucleotide, preferably 2'-O-methyl, 2'-
deozy-2'-
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fluoro, or 2'-OH; U is a nucleotide, preferably 2'-O-methyl, 2'-deozy-2'-
fluoro, or 2'-OH;
C represents a nucleotide, preferably 2'-amino (e.g., 2'-NH2 or 2'-O- NH2, and
represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage,
phosphorothioate, phosphorodithioate or others known in the art).
The enzymatic nucleic acid molecules of Formula 4 and Formula 5 may
independently comprise a cap structure which may independently be present or
absent.
In yet another preferred embodiment, the 3'-cap is selected from a group
comprising,
4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio
nucleotide;
carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl
phosphate; 3-
aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate;
hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-
nucleotide;
modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide;
acyclic
3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl
nucleotide; 5'-5'-
inverted nucleotide moiety; S'-5'-inverted abasic moiety; S'-phosphoramidate;
5'-
phosphorothioate; 1,4-butanediol phosphate 5'-amino; bridging and/or non-
bridging 5'-
phosphoramidate, phosphorothioate and/or hosphorodithioate; bridging or non
bridging
methylphosphonate and S'-mercapto moieties (for more details, see Beaucage and
Iyer,
1993, Tetrahedron 49, 1925; incorporated by reference herein).
In another aspect, the invention provides mammalian cells containing one or
more
nucleic acid molecules and/or expression vectors of this invention. The one or
more
nucleic acid molecules may independently be targeted to the same or different
sites.
Nucleotide Synthesis
Addition of dimethylaminopyridine (DMAP) to the phosphorylation protocols
known in the art can greatly increase the yield of nucleotide monophosphates
while
decreasing the reaction time. Synthesis of the nucleosides of the invention
have been
described in several publications and Applicants previous applications
(Beigelman et al.,
International PCT publication No. WO 96/18736; Dudzcy et al., Int. PCT Pub.
No. WO
95/11910; Usman et al., Int. PCT Pub. No. WO 95/13378; Matulic-Adamic et al.,
1997,
Tetrahedron Lett. 38, 203; Matulic-Adamic et al., 1997, Tetrahedron Lett. 38,
1669; all of
which are incorporated herein by reference). These nucleosides are dissolved
in triethyl
phosphate and chilled in an ice bath. Phosphorus oxychloride (POC13) is then
added
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followed by the introduction of DMAP. The reaction is then warmed to room
temperature
and allowed to proceed for S hours. This reaction allows the formation of
nucleotide
monophosphates which can then be used in the formation of nucleotide
triphosphates.
Tributylamine is added followed by the addition of anhydrous acetonitrile and
tributylammonium pyrophosphate. The reaction is then quenched with TEAB and
stirred
overnight at room temperature (about 20°C). The triphosphate is
purified using
Sephadex~ column purification or equivalent and/or HPLC and the chemical
structure is
confirmed using NMR analysis. Those skilled in the art will recognize that the
reagents,
temperatures of the reaction, and purification methods can easily be
alternated with
substitutes and equivalents and still obtain the desired product.
Nucleotide Tri~hosphates
The invention provides nucleotide triphosphates which can be used for a number
of
different functions. The nucleotide triphosphates formed from nucleosides
found in Table
45 are unique and distinct from other nucleotide triphosphates known in the
art.
Incorporation of modified nucleotides into DNA or RNA oligonucleotides can
alter the
properties of the molecule. For example, modified nucleotides can hinder
binding of
nucleases, thus increasing the chemical half life of the molecule. This is
especially
important if the molecule is to be used for cell culture or in vivo. It is
known in the art that
the introduction of modified nucleotides into these molecules can greatly
increase the
stability and thereby the effectiveness of the molecules (Burgin et al., 1996,
Biochemistry
35, 14090-14097; Usman et al., 1996, Curr. Opin. Struct. Biol. 6, 527-533).
Modified nucleotides are incorporated using either wild type or mutant
polymerases.
For example, mutant T7 polymerase is used in the presence of modified
nucleotide
triphosphate(s), DNA template and suitable buffers. Those skilled in the art
will recognize
that other polymerases and their respective mutant versions can also be
utilized for the
incorporation of NTP's of the invention. Nucleic acid transcripts were
detected by
incorporating radiolabelled nucleotides (a-32P NTP). The radiolabeled NTP
contained the
same base as the modified triphosphate being tested. The effects of methanol,
PEG and
LiCI were tested by adding these compounds independently or in combination.
Detection
and quantitation of the nucleic acid transcripts was performed using a
Molecular Dynamics
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PhosphorImager. Efficiency of transcription was assessed by comparing modified
nucleotide triphosphate incorporation with all-ribonucleotide incorporation
control. Wild-
type polymerise was used to incorporate NTP-'s using the manufacturer's
buffers and
instructions (Boehringer Mannheim).
Transcription Conditions
Incorporation rates of modified nucleotide triphosphates into oligonucleotides
can be
increased by adding to traditional buffer conditions, several different
enhancers of
modified NTP incorporation. Applicant has utilized methanol and LiCI in an
attempt to
increase incorporation rates of dNTP using RNA polymerise. These enhancers of
modified NTP incorporation can be used in different combinations and ratios to
optimize
transcription. Optimal reaction conditions differ between nucleotide
triphosphates and can
readily be determined by standard experimentation. Overall, however, Applicant
has
found that inclusion of enhancers of modified NTP incorporation such as
methanol or
inorganic compound such as lithium chloride increase the mean transcription
rates.
Applicant synthesized pyrimidine nucleotide triphosphates using DMAP in the
reaction. For purines, applicant utilized standard protocols previously
described in the art
(Yoshikawa et al supra; . Ludwig, supra). Described below is one example of a
pyrimdine
nucleotide triphosphate and one purine nucleotide triphosphate synthesis.
Synthesis of purine nucleotide triphosphates: 2'-O-methyl-~uanosine-5'-
triphosphate
2'-O-methyl guanosine nucleoside (0.25 grams, 0.84 mmol) was dissolved in
triethyl
phosphate (5.0) ml by heating to 100°C for 5 minutes. The resulting
clear, colorless
solution was cooled to 0°C using an ice bath under an argon atmosphere.
Phosphorous
oxychloride (1.8 eq., 0.141 ml) was then added to the reaction mixture with
vigorous
stirring. The reaction was monitored by HPLC, using a sodium perchlorate
gradient.
After 5 hours at 0°C, tributylamine (0.65 ml) was added followed by the
addition of
anhydrous acetonitrile (10.0 ml), and after S minutes (reequilibration to
0°C)
tributylammonium pyrophosphate (4.0 eq., 1.53 g) was added. The reaction
mixture was
quenched with 20 ml of 2M TEAB after 15 minutes at 0°C (HPLC analysis
with above
conditions showed consumption of monophosphate at 10 minutes) then stirred
overnight at
room temperature, the mixture was evaporated in vacuo with methanol co-
evaporation
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(4x) then diluted in 50 ml O.OSM TEAB. DEAF sephadex purification was used
with a
gradient of 0.05 to 0.6 M TEAB to obtain pure triphosphate (0.52 g, 66.0%
yield) (elutes
around 0.3M TEAB); the purity was confirmed by HPLC and NMR analysis.
Smthesis of Pyrimidine nucleotide triphosphates: 2'-O-methylthiomethyl-uridine-
5'-
trinhosphate
2'-O-methylthiomethyl uridine nucleoside (0.27 grams, 1.0 mmol) was dissolved
in
triethyl phosphate (5.0 ml). The resulting clear, colorless solution was
cooled to 0°C with
an ice bath under an argon atmosphere. Phosphorus oxychloride (2.0 eq., 0.190
ml) was
then added to the reaction mixture with vigorous stirring.
Dimethylaminopyridine
(DMAP, 0.2eq., 25 mg) was added, the solution warmed to room temperature and
the
reaction was monitored by HPLC, using a sodium perchlorate gradient. After 5
hours at
20°C, tributylamine (1.0 ml) was added followed by anhydrous
acetonitrile (10.0 ml), and
after 5 minutes tributylammonium pyrophosphate (4.0 eq., 1.8 g) was added. The
reaction
mixture was quenched with 20 ml of 2M TEAB after 15 minutes at 20°C
(HPLC analysis
with above conditions showed consumption of monophosphate at 10 minutes) then
stirred
overnight at room temperature. The mixture was evaporated in vacuo with
methanol co-
evaporation (4x) then diluted in SO ml O.OSM TEAB. DEAF fast flow Sepharose
purification with a gradient of 0.05 to 1.0 M TEAB was used to obtain pure
triphosphate
(0.40 g, 44% yield) (elutes around 0.3M TEAB) as determined by HPLC and NMR
analysis.
Utilization of DMAP in Uridine 5'-Triphosphate Synthesis
The reactions were performed on 20 mg aliquots of nucleoside dissolved in 1 ml
of
triethyl phosphate and 19 u1 of phosphorus oxychloride. The reactions were
monitored at
40 minute intervals automatically by HPLC to generate yield-of product curves
at times up
to 18 hours. A reverse phase column and ammonium acetate/ sodium acetate
buffer
system (SOmM & 100mM respectively at pH 4.2) was used to separate the 5', 3',
2'
monophosphates (the monophosphates elute in that order) from the 5'-
triphosphate and the
starting nucleoside. The data is shown in Table 46. These conditions doubled
the product
yield and resulted in a 10-fold improvement in the reaction time to maximum
yield (1200
minutes down to 120 minutes for a 90% yield). Selectivity for 5'-
monophosphorylation
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was observed for all reactions. Subsequent triphosphorylation occurred in
nearly
quantitative yield.
Materials Used in Bacteriopha~e T7 RNA Polymerase Reactions
Buffer 1: Reagents are mixed together to form a l OX stock solution of buffer
1
(400 mM Tris-Cl [pH 8.1], 200 mM MgCl2, 100 mM DTT, 50 mM spermidine, and 0.1%
triton~ X-100). Prior to initiation of the polymerise reaction methanol, LiCI
is added and
the buffer is diluted such that the final reaction conditions for condition 1
consisted of
40mM tris (pH 8.1), 20mM MgCl2, 10 mM DTT, 5 mM spermidine, 0.01% triton~ X-
100, 10% methanol, and 1 mM LiCI.
BUFFER 2: Reagents are mixed together to form a l OX stock solution of buffer
2
(400 mM Tris-Cl [pH 8.1 ], 200 mM MgCl2, 100 mM DTT, 50 mM spermidine, and 0.1
triton~ X-100). Prior to initiation of the polymerise reaction PEG, LiCI is
added and the
buffer is diluted such that the final reaction conditions for buffer 2
consisted of : 40mM
tris (pH 8.1), 20mM MgCl2, 10 mM DTT, 5 mM spermidine, 0.01% triton~ X-100, 4%
PEG, and 1 mM LiCI.
BUFFER 3: Reagents are mixed together to form a l OX stock solution of buffer
3
(400 mM Tris-Cl [pH 8.0], 120 mM MgCl2, 50 mM DTT, 10 mM spermidine and 0.02%
triton~ X-100). Prior to initiation of the polymerise reaction PEG is added
and the buffer
is diluted such that the final reaction conditions for buffer 3 consisted of :
40mM tris (pH
8.0), 12 mM MgCl2, 5 mM DTT, 1 mM spermidine, 0.002% triton~ X-100, and 4%
PEG.
BUFFER 4: Reagents are mixed together to form a l OX stock solution of buffer
4
(400 mM Tris-Cl [pH 8.0], 120 mM MgCl2, 50 mM DTT, 10 mM spermidine and 0.02%
triton~ X-100). Prior to initiation of the polymerise reaction PEG, methanol
is added and
the buffer is diluted such that the final reaction conditions for buffer 4
consisted of
40mM tris (pH 8.0), 12 mM MgCl2, 5 mM DTT, 1 mM spermidine, 0.002% triton~ X-
100, 10% methanol, and 4% PEG.
BUFFER 5: Reagents are mixed together to form a l OX stock solution of buffer
5
(400 mM Tris-Cl [pH 8.0], 120 mM MgCl2, 50 mM DTT, 10 mM spermidine and 0.02%
triton~ X-100). Prior to initiation of the polymerise reaction PEG, LiCI is
added and the
buffer is diluted such that the final reaction conditions for buffer 5
consisted of : 40mM
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tris (pH 8.0), 12 mM MgCl2, 5 mM DTT, 1 mM spermidine, 0.002% triton~ X-100, 1
mM LiCI and 4% PEG.
BUFFER 6: Reagents are mixed together to form a l OX stock solution of buffer
6
(400 mM Tris-Cl [pH 8.0], 120 mM MgCl2, SO mM DTT, 10 mM spermidine and 0.02%
triton~ X-100). Prior to initiation of the polymerise reaction PEG, methanol
is added and
the buffer is diluted such that the final reaction conditions for buffer 6
consisted of
40mM tris (pH 8.0), 12 mM MgCl2, 5 mM DTT, 1 mM spermidine, 0.002% triton~ X-
100, 10% methanol, and 4% PEG.
BUFFER 7: Reagents are mixed together to form a l OX stock solution of buffer
6
(400 mM Tris-Cl [pH 8.0], 120 mM MgCl2, 50 mM DTT, 10 mM spermidine and 0.02%
triton~ X-100). Prior to initiation of the polymerise reaction PEG, methanol
and LiCI is
added and the buffer is diluted such that the final reaction conditions for
buffer 6 consisted
of : 40mM tris (pH 8.0), 12 mM MgCl2, 5 mM DTT, 1 mM spermidine, 0.002%
triton~
X-100, 10% methanol, 4% PEG, and 1 mM LiCI.
Screening of Modified nucleotide triphosphates with Mutant T7 RNA Polymerise
Modified nucleotide triphosphates were tested in buffers 1 through 6 at two
different
temperatures (25 and 37°C). Buffers 1-6 tested at 25°C were
designated conditions 1-6
and buffers 1-6 tested at 37°C were designated conditions 7-12 (Table
47). In each
condition, Y639F mutant T7 polymerise (Sousa and Padilla, supra) (0.3-2 mg/20
ml
reaction), NTP's (2 mM each), DNA template (10 pmol), inorganic
pyrophosphatase
(SU/ml) and oc-32P NTP (0.8 mCi/pmol template) were combined and heated at the
designated temperatures for 1-2 hours. The radiolabeled NTP used was different
from the
modified triphosphate being testing. The samples were resolved by
polyacrylamide gel
electrophoresis. Using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA),
the
amount of full-length transcript was quantified and compared with an all-RNA
control
reaction. The data is presented in Table 48; results in each reaction are
expressed as a
percent compared to the all-ribonucleotide triphosphate (rNTP) control. The
control was
run with the mutant T7 polymerise using commercially available polymerise
buffer
(Boehringer Mannheim, Indianapolis,1N).
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Incorporation of Modified NTP's using Wild-type T7 RNA polymerise
Bacteriophage T7 RNA polymerise was purchased from Boehringer Mannheim at
0.4 U/pL concentration. Applicant used the commercial buffer supplied with the
enzyme
and 0.2 pCi alpha-32P NTP in a 50 pL reaction with nucleotides triphosphates
at 2 mM
each. The template was a double-stranded PCR fragment, which was used in
previous
screens. Reactions were carned out at 37°C for 1 hour. Ten pL of the
sample was run on
a 7.5% analytical PAGE and bands were quantitated using a PhosphorImager.
Results are
calculated as a comparison to an "all ribo" control (non-modified nucleotide
triphosphates)
and the results are in Table 49.
Incorporation of Multiple Modified nucleotide triphosphates Into
Oli~onucleotides
Combinations of modified nucleotide triphosphates were tested with the
transcription protocol described above, to determine the rates of
incorporation of two or
more of these triphosphates. Incorporation of 2'-Deoxy-2'-(L-histidine) amino
uridine (2'-
his-NH2-UTP) was tested with unmodified cytidine nucleotide triphosphates,
rATP and
rGTP in reaction condition number 9. The data is presented as a percentage of
incorporation of modified NTP's compared to the all rNTP control and is shown
in Table
50a.
Two modified cytidines (2'-NHZ-CTP or 2'dCTP) were incorporated along with 2'-
his-NHZ-UTP with identical efficiencies. 2'-his-NH2-UTP and 2'-NHZ-CTP were
then
tested with various unmodified and modified adenosine triphosphates in the
same buffer
(Table 50b). The best modified adenosine triphosphate for incorporation with
both 2'-his-
NHZ-UTP and 2'-NHZ-CTP was 2'-NHZ-DAPTP.
Outimization of Reaction conditions for Incorporation of Modified Nucleotide
Triphosphate
The combination of 2'-his-NHZ-UTP, 2'-NH2-CTP, 2'-NHZ-DAP, and rGTP was
tested in several reaction conditions (Table 51) using the incorporation
protocol described
above. The results demonstrate that of the buffer conditions tested,
incorporation of these
modified nucleotide triphosphates occur in the presence of both methanol and
LiCI.
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Selection of Novel Enzymatic nucleic acid molecule Motifs using 2'-deoxv-
2'amino
Modified GTP and CTP
For selection of new enzymatic nucleic acid molecule motifs, pools of
enzymatic
nucleic acid molecules were designed to have two substrate binding arms (5 and
16
nucleotides long) and a random region in the middle. The substrate has a
biotin on the 5'
end, S nucleotides complementary to the short binding arm of the pool, an
unpaired G (the
desired cleavage site), and 16 nucleotides complementary to the long binding
arm of the
pool. The substrate was bound to column resin through an avidin-biotin
complex. The
general process for selection is shown in Figure 11. The protocols described
below
represent one possible method that may be utilized for selection of enzymatic
nucleic acid
molecules and are given as a non-limiting example of enzymatic nucleic acid
molecule
selection with combinatorial libraries.
Construction of Libraries:
The oligonucleotides listed below were synthesized by Operon Technologies
(Alameda, CA). Templates were gel purified and then run through a Sep-PakTM
cartridge
(Waters, Millford, MA) using the manufacturers protocol. Primers (MST3, MST7c,
MST3del) were used without purification.
Primers:
MST3 (30 mer): 5'- CAC TTA GCA TTA ACC CTC ACT AAA GGC CGT-3'
MST7c (33 mer): S'-TAA TAC GAC TCA CTA TAG GAA AGG TGT GCA ACC-3'
MST3del (18 mer): 5'-ACC CTC ACT AAA GGC CGT-3'
Templates:
MSN60c (93 mer): 5'-ACC CTC ACT AAA GGC CGT (I~6o GGT TGC ACA CCT
TTG-3'
MSN40c (73 mer): 5'-ACC CTC ACT AAA GGC CGT (N'4o GGT TGC ACA CCT
TTG-3'
MSN20c (53 mer): 5'-ACC CTC ACT AAA GGC CGT (1~2o GGT TGC ACA CCT
TTG-3'
N60 library was constructed using MSN60c as a template and MST3/MST7c as
primers. N40 and N20 libraries were constructed using MSN40c (or MSN20c) as
template
and MST3de1/MST7c as primers.
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Single-stranded templates were converted into double-stranded DNA by the
following protocol: 5 nmol template, 10 nmol each primer, in 10 ml reaction
volume
using standard PCR buffer, dNTP's, and taq DNA polymerase (all reagents from
Boerhinger Mannheim). Synthesis cycle conditions were 94°C, 4 minutes;
(94°C, 1
minute; 42°C, 1 minute; 72°C, 2 minutes) x 4; 72°C, 10
minutes. Products were
checked on agarose gel to confirm the length of each fragment (N60=123 bp,
N40=91 bp,
N20=71 bp) and then were phenol/chloroform extracted and ethanol precipitated.
The
concentration of the double-stranded product was 25 wM.
Transcription of the initial pools was performed in a 1 ml volume comprising:
500
pmol double-stranded template (3 x 10'4 molecules), 40 mM tris-HCl (pH 8.0),
12 mM
MgCl2, 1 mM spermidine, 5 mM DTT, 0.002% triton X-100, 1 mM LiCI, 4% PEG 8000,
10% methanol, 2 mM ATP (Pharmacia), 2 mM GTP (Pharmacia), 2 mM 2'-deoxy-2'-
amino-CTP (USB), 2 mM 2'-deoxy-2'-amino-UTP (LTSB), 5 U/ml inorganic
pyrophosphatase (Sigma), S U/pl T7 RNA polymerase (USB; Y639F mutant was used
in
some cases at 0.1 mg/ml (Sousa and Padilla, supra)), 37°C, 2 hours.
Transcribed libraries
were purified by denaturing PAGE (N60=106 ntds, N40=74, N20=54) and the
resulting
product was desalted using Sep-PakTM columns and then ethanol precipitated.
Initial column-Selection:
The following biotinylated substrate was synthesized using standard protocols
(Usman et al., 1987 J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990
Nucleic Acids
Res., 18, 5433; and Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684):
5'-biotin-C18 spacer-GCC GUG GGU UGC ACA CCU UUC C-C18 spacer-thiol-
modifier C6 S-S-inverted abasic-3'
Substrate was purified by denaturing PAGE and ethanol precipitated. 10 nmol of
substrate was linked to a NeutrAvidinTM column using the following protocol:
400 p1
UltraLink Immobilized NeutrAvidinTM slurry (200 ~.1 beads, Pierce, Rockford,
IL) were
loaded into a polystyrene column (Pierce). The column was washed twice with 1
ml of
binding buffer (20 mM NaP04 (pH 7.5), 150 mM NaCI) and then capped off (i.e.,
a cap
was put on the bottom of the column to stop the flow). 200 p1 of the substrate
suspended
in binding buffer was applied and allowed to incubate at room temperature for
30 minutes
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with occasional vortexing to ensure even linking and distribution of the
solution to the
resin. After the incubation, the cap was removed and the column was washed
with 1 ml
binding buffer followed by 1 ml column buffer (50 mM tris-HCL (pH 8.5), 100 mM
NaCI,
50 mM KCl). The column was then ready for use and capped off. 1 nmol of the
initial
pool RNA was loaded on the column in a volume of 200 ~1 column buffer. It was
allowed
to bind the substrate by incubating for 30 minutes at room temperature with
occasional
vortexing. After the incubation, the cap was removed and the column was washed
twice
with 1 ml column buffer and capped off. 200 p1 of elution buffer (50 mM tris-
HCl (pH
8.5), 100 mM NaCI, 50 mM KCI, 25 mM MgCl2) was applied to the column followed
by
30 minute incubation at room temperature with occasional vortexing. The cap
was
removed and four 200 p1 fractions were collected using elution buffer.
Second column (counter selection):
A diagram for events in the second column is generally shown in Figure 12 and
substrate oligonucleotide used is shown below:
5'-GGU UGC ACA CCU UUC C-C18 spacer-biotin-inverted abasic-3'
This column substrate was linked to UltraLink NeutrAvidinTM resin as
previously
described (40 pmol) which was washed twice with elution buffer. The eluent
from the
first column purification was then run on the second column. The use of this
column
allowed for binding of RNA that non-specifically diluted from the first
column, while
RNA that performed a catalytic event and had product bound to it, flowed
through the
second column. The fractions were ethanol precipitated using glycogen as
carrier and
rehydrated in sterile water for amplification.
Amplification:
RNA and primer MST3 (10-100 pmol) were denatured at 90°C for 3
minutes in
water and then snap-cooled on ice for one minute. The following reagents were
added to
the tube (final concentrations given): 1X PCR buffer (Boerhinger Mannheim), 1
mM
dNTP's (for PCR, Boerhinger Mannheim), 2 U/~1 RNase-Inhibitor (Boerhinger
Mannheim), 10 U/pl SuperscriptTM II Reverse Transcriptase (BRL). The reaction
was
incubated for 1 hour at 42°C, then at 95°C for 5 minutes in
order to destroy the
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SuperscriptTM. The following reagents were then added to the tube to increase
the volume
five-fold for the PCR step (final concentrations/amounts given): MST7c primer
(10-100
pmol, same amount as in RT step), 1X PCR buffer, taq DNA polymerase (0.025-
0.05
U/~1, Boerhinger Mannheim). The reaction was cycled as follows: 94°C,
4minutes;
(94°C, 30s; 42-54°C, 30s; 72°C, lminute) x 4-30 cycles;
72°C, Sminutes; 30°C, 30
minutes. Cycle number and annealing temperature were decided on a round by
round
basis. In cases where heteroduplex was observed, the reaction was diluted five-
fold with
fresh reagents and allowed to progress through 2 more amplification cycles.
Resulting
products were analyzed for size on an agarose gel (N60=123 bp, N40=103 bp,
N20=83 bp)
and then ethanol precipitated.
Transcriptions:
Transcription of amplified products was done using the conditions described
above
with the following modifications: 10-20% of the amplification reaction was
used as
template, reaction volume was 100-500 p1, and the products sizes varied
slightly
(N60=106 ntds, N40=86, N20=66). A small amount of 32P-GTP was added to the
reactions for quantitation purposes.
Subsequent rounds:
Subsequent rounds of selection used 20 pmols of input RNA and 40 pmol of the
22
nucleotide substrate on the column.
Activity of pools:
Pools were assayed for activity under single turnover conditions every three
to four
rounds. Activity assay conditions were as follows: 50 mM tris-HCl (pH 8.5), 25
mM
MgCl2, 100 mM NaCI, 50 mM KCI, trace 32P-labeled substrate, 10 nM RNA pool. 2X
pool in buffer and, separately, 2X substrate in buffer were incubated at
90°C for 3 minutes,
then at 37°C for 3 minutes. Equal volume 2X substrate was then added
the 2X pool tube
(t=0). Initial assay time points were taken at 4 and 24 hours: 5 ~.1 was
removed and
quenched in 8 ~1 cold Stop buffer (96% formamide, 20 mM EDTA, 0.05% bromphenyl
blue/xylene cyanol). Samples were heated 90°C, 3 minutes, and loaded on
a 20%
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sequencing gel. Quantitation was performed using a Molecular Dynamics
Phosphorimager
and ImageQuaNTTM software. The data is shown in Table 52.
Samples from the pools of oligonucleotide were cloned into vectors and
sequenced
using standard protocols (Sambrook et al., Molecular Cloning: A Laboratory
Manual,
Cold Spring Harbor Laboratory Press). The enzymatic nucleic acid molecules
were
transcribed from a representative number of these clones using methods
described in this
application. Individuals from each pool were tested for RNA cleavage from N60
and N40
by incubating the enzymatic nucleic acid molecules from the clones with S/16
substrate in
2mM MgCl2, pH 7.5, l OmM KCl at 37°C. The data in Table 54 shows that
the enzymatic
nucleic acid molecules isolated from the pool are individually active.
Kinetic Activity:
Kinetic activity of the enzymatic nucleic acid molecule shown in Table 54, was
determined by incubating enzymatic nucleic acid molecule (10 nM) with
substrate in a
cleavage buffer (pH 8.5, 25 mM MgClz, 100 mM NaCI, 50 mM KC1) at 37°C.
Magnnesium Dependence:
Magnesium dependence of round 15 of N20 was tested by varying MgCl2 while
other conditions were held constant (50 mM tris [pH 8.0], 100 mM NaCI, 50 mM
KCI,
single turnover, 10 nM pool). The data is shown in Table 55, which
demonstrates
increased activity with increased magnesium concentrations.
Selection of Novel Enzymatic nucleic acid molecule Motifs using 2'-Deoxy-2'-(N
histidyl~ amino UTP, 2'-Fluoro-ATP, and 2'-deoxy-2'-amino CTP and GTP
The method used for selection of novel enzymatic nucleic acid molecule motifs
using 2'-deoxy-2'amino modified GTP and CTP was repeated using 2'-Deoxy-2'-(N
histidyl) amino UTP, 2'-Fluoro-ATP, and 2'-deoxy-2'-amino CTP and GTP.
However,
rather than causing cleavage on the initial column with MgClz, the initial
random
modified-RNA pool was loaded onto substrate-resin in the following buffer; 5
mM
NaOAc pH 5.2, 1 M NaCI at 4° C. After ample washing, the resin was
moved to 22 ° C
and the buffer switch 20 mM HEPES pH 7.4, 140 mM KCI, 10 mM NaCI, 1 mM CaCl2,
1 mM MgCl2. In one selection of N60 oligonucleotides, no divalent canons
(MgCl2,
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CaCl2) was used. The resin was incubated for 10 minutes to allow reaction and
the eluant
collected.
The enzymatic nucleic acid molecule pools were capable of cleaving 1-3% of the
present substrate even in the absence of divalent cations, the background (in
the absence of
modified pools) was 0.2 - 0.4 %.
Synthesis of 5-substituted 2'-modified nucleosides
When designing monomeric nucleoside triphosphates for selection of therapeutic
catalytic RNAs, one has to take into account nuclease stability of such
molecules in
biological sera. A common approach to increase RNA stability is to replace the
sugar 2'-
OH group with other groups like 2'-fluoro, 2'-O-methyl or 2'-amino.
Fortunately such 2'-
modified pyrimidine 5'triphosphates are shown to be substrates for RNA
polymerases.
(Aurup, H.; Williams, D.M.; Eckstein, F. Biochemistry 1992, 31, 9637; and
Padilla, R.;
Sousa, R. Nucleic Acids Res. 1999, 27, 1561.) On the other hand it was shown
that
variety of substituents at pyrimidine 5-position is well tolerated by T7 RNA
polymerase
(Tarasow, T.M.; Eaton, B.E. Biopolymers 1998, 48, 29), most likely because the
natural
hydrogen-bonding pattern of these nucleotides is preserved. We have chosen 2'-
fluoro and
2'-D-methyl pyrimidine nucleosides as starting materials for attachment of
different
functionalities to the 5-position of the base. Both rigid (alkynyl) and
flexible (alkyl)
spacers are used. The choice of imidazole, amino and carboxylate pendant
groups is based
on their ability to act as general acids, general bases, nucleophiles and
metal ligands, all of
which can improve the catalytic effectiveness of selected nucleic acids.
Figures 21 - 24
relate to the synthesis of these compounds.
2'-O-methyluridine was 3',5'-bis-acetylated using acetic anhydride in pyridine
and
then converted to its 5-iodo derivative 1 a using I2/ceric ammonium nitrate
reagent
(Asakura, J.; Robins, M.J. J. Org. Chem. 1990, 55, 4928) (Scheme 1). Both
reactions
proceeded in a quantitative yield and no chromatographic purifications were
needed.
Coupling between 1 and N trifluoroacetyl propargylamine using copper(I) iodide
and
tetrakis(triphenylphosphine)palladium(0) catalyst as described by Hobbs
(Hobbs, F.W., Jr.
J. Org. Chem. 1989, 54, 3420) yielded 2a in 89% yield. Selective O-deacylation
with
aqueous NaOH afforded 3a which was phosphorylated with POCl3/triethylphosphate
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(TEP) in the presence of 1,8-bis(dimethylamino)naphthalene (Proton-Sponge)
(Method A)
(Kovacz, T; Otvos, L. Tetrahedron Lett. 1988, 29, 4525). The intermediate
nucleoside
phosphorodichloridate was condensed in situ with tri-n-butylammonium
pyrophosphate.
At the end, the N TFA group was removed with concentrated ammonia. 5'-
Triphosphate
was purified on Sephadex~ DEAF A-25 ion exchange column using a linear
gradient of
0.1-0.8M triethylammonium bicarbonate (TEAB) for elution. Traces of
contaminating
inorganic pyrophosphate are removed using C-18 RP HPLC to afford analytically
pure
material. Conversion into Na-salt was achieved by passing the aqueous solution
of
triphosphate through Dowex SOWX8 ion exchange resin in Na+ form to afford 4a
in 45%
yield. When Proton-Sponge was omitted in the first phosphorylation step,
yields were
reduced to 10-20%. Catalytic hydrogenation of 3a yielded S-aminopropyl
derivative Sa
which was phosphorylated under conditions identical to those described for
propynyl
derivative 3a to afford triphosphate 6a in SO% yield.
For the preparation of imidazole derivatized triphosphates 9a and 11a, we
developed an efficient synthesis of N diphenylcarbamoyl 4-imidazoleacetic acid
(~DPC): Transient protection of carboxyl group as TMS-ester using TMS
Cl/pyridine followed by DPC-Cl allowed for a clean and quantitative conversion
of 4
imidazoleacetic acid (ImAA) to its N DPC protected derivative.
Complete deacylation of 2a afforded 5-(3-aminopropynyl) derivative 8a which
was condensed with 4-imidazoleacetic acid in the presence of 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide (EDC) to afford 9a in 68% yield.
Catalytic
hydrogenation of 8a yielded S-(3-aminopropyl) derivative 10a which was
condensed
with ImAADP~ to yield conjugate lla in 32% yield. Yields in these couplings
were
greatly improved when S'-OH was protected with DMT group (not shown) thus
efficiently preventing undesired 5'-O-esterification. Both 9a and 11 a failed
to yield
triphosphate products in reaction with POC13/TEP/Proton-Sponge.
On the contrary, phosphorylation of 3'-O-acetylated derivatives 12a and 13a
using
2-chloro-4H 1,3,2-benzodioxaphosphorin-4-one followed by pyrophosphate
addition
and oxidation (Method B, Scheme 2; Ludwig, J., Eckstein, F., J. Org. Chem.
1989, 54,
631) afforded the desired triphosphates 14a and 15a in 57% yield,
respectively.
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2'-Deoxy-2'-fluoro nucleoside 5'-triphosphates containing amino- (4b, 6b) and
imidazole- (14b,15b) linked groups were synthesized in a manner analogous to
that
described for the preparation of 2'-O-methyl nucleoside 5'-triphosphates
(Schemes 1 and
2). Again, only Ludwig-Eckstein's phosphorylation worked for the preparation
of 4-
imidazoleacetyl derivatized triphosphates.
It is worth noting that when "one-pot-two-steps" phosphorylation reaction
(Kovacz, T; Otvos, L. Tetrahedron Lett. 1988, 29, 4525) of 5b was quenched
with 40%
aqueous methylamine instead of TEAB or H20, the y-amidate 7b was generated as
the
only detectable product. Similar reaction was reported recently for the
preparation of the
y-amidate of pppA2'p5'A2'p5'A.12
Carboxylate group was introduced into 5-position of uridine both on the
nucleoside
level and post-synthetically (Method C) (Scheme 3). 5-Iodo-2'-deoxy-2'-
fluorouridine (16)
was coupled with methyl acrylate using modified Heck reactionl3 to yield 17 in
85% yield.
5'-O-Dimethoxytritylation, followed by in situ 3'-O-acetylation and subsequent
detritylation afforded 3'-protected derivative 18. Phosphorylation using 2-
chloro-4H
1,3,2-benzodioxa-phosphorin-4-one followed by pyrophosphate addition and
oxidation
(Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631) afforded the desired
triphosphate
in 54% yield. On the other hand, 5-(3-aminopropyl)uridine 5'-triphosphate 6b
was coupled
with N hydroxysuccinimide ester of Fmoc-Asp-OFm to afford, after removal of
Fmoc and
Fm groups with diethylamine, the desired aminoacyl conjugate 20 in 50% yield.
Cytidine derivatives comprising 3-aminopropyl and 3(N succinyl)aminopropyl
groups were synthesized according to Scheme 4. Peracylated 5-(3-
aminopropynyl)uracil
derivative 2b is reduced using catalytic hydrogenation and then converted in
seven steps
and S% overall yield into 3'-acetylated cytidine derivative 25. This synthesis
was plagued
by poor solubility of intermediates and formation of the N4-cyclized byproduct
during
ammonia treatment of the 4-triazolyl intermediate. Phosphorylation of 25 as
described in
reference 11 yielded triphosphate 26 and N4-cyclized product 27 in 1:1 ratio.
They were
easily separated on Sephadex DEAF A-25 ion exchange column using 0.1-0.8M TEAB
gradient. It appears that under basic conditions the free primary amine can
displace any
remaining intact 4-NHBz group leading to the cyclized product. This is similar
to
displacement of 4-triazolyl group by primary amine as mentioned above.
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We reasoned that utilization of N4-unprotected cytidine will solve this
problem.
This lead to an improved synthesis of 26: Iodination of 2'-deoxy-2'-
fluorocytidine (28)
provided the S-iodo derivative 29 in 58% yield. This compound was then
smoothly
converted into S-(3-aminopropynyl) derivative 30. Hydrogenation afforded 5-(3-
aminopropyl) derivative 31 which was phosphorylated directly with POC13/ PPi
to afford
26 in 37% yield. Coupling of the 5'-triphosphate 26 with succinic anhydride
yielded
succinylated derivative 32 in 36% yield.
Synthesis of 5-Imidazoleacetic acid 2'-deoxy-5'-triphosphate uridine
5-dintrophenylimidazoleacetic acid 2'-deoxy uridine nucleoside (80 mg) was
dissolved in 5 ml of triethylphosphate while stirnng under argon, and the
reaction mixture
was cooled to 0°C. Phosphorous oxychloride (1.8 eq, 22 ml) was added to
the reaction
mixture at 0°C, three more aliquots were added over the course of 48
hours at room
temperature. The reaction mixture was then diluted with anhydrous MeCN (5 ml)
and
cooled to 0°C, followed by the addition of tributylamine (0.65 ml) and
tributylammonium
pyrophosphate (4.0 eq, 0.24 g). After 45 minutes, the reaction was quenched
with 10 ml
aq. methyl amine for four hours. After co-evaporation with MeOH (3x), purified
material
on DEAF Sephadex followed by RP chromatography to afford 15 mg of
triphosphate.
Synthesis of 2'-(N lysyll-amino-2'-deoxy-cytidine Triphosphate
2'-(N-lysyl)-amino-2'-deoxy cytidine (0.180 g, 0.22 mmol) was dissolved in
triethyl
phosphate (2.00 ml) under Ar. The solution was cooled to 0 °C in an ice
bath.
Phosphorus oxychloride (99.999%, 3 eq., 0.0672 mL) was added to the solution
and the
reaction was stirred for two hours at 0 °C. Tributylammonium
pyrophosphate (4 eq., 0.400
g) was dissolved in 3.42 mL of acetonitrile and tribuytylamine (0.165 mL).
Acetonitrile (1
mL) was added to the monophosphate solution followed by the pyrophosphate
solution
which was added dropwise. The resulting solution was clear. The reaction was
allowed to
warm up to room temperature. After stirring for 45 minutes, methylamine (5 mL)
was
added and the reaction and stirred at room temperature for 2 hours. A biphasic
mixture
appeared (little beads at the bottom of the flask). TLC (7:1:2
iPrOH:NH40H:H20)
showed the appearance of triphosphate material. The solution was concentrated,
dissolved
in water and loaded on a newly prepared DEAF Sephadex A-25 column. The column
was
washed with a gradient up to 0.6 M TEAB buffer and the product eluted off in
fractions
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90-95. The fractions were analyzed by ion exchange HPLC. Each fraction showed
one
triphosphate peak that eluted at 4.000 minutes. The fractions were combined
and
pumped down from methanol to remove buffer salt to yield 15.7 mg of product.
thesis of 2'-deoxy-2'-(L-histidine)amino Cytidine Triphosphate
2'-[N Fmoc, lVimid _dinitrophenyl-histidyl]amino-2'-cytidine (0.310 g, 4.04
mmol)
was dissolved in triethyl phosphate (3 ml) under Ar. The solution was cooled
to 0 °C.
Phosphorus oxychloride (1.8 eq., 0.068 mL) was added to the solution and
stored
overnight in the freezer. The next morning TLC (10% MeOH in CH2C12) showed
significant starting material, one more equivalent of POC13 was added. After
two hours,
TLC still showed starting material. Tributylamine (0.303 mL) and
Tributylammonium
pyrophosphate (4 eq., 0.734 g) dissolved in 6.3 mL of acetonitrile (added
dropwise) were
added to the monophosphate solution. The reaction was allowed to warm up to
room
temperature. After stirring for 15 min, methylamine (10 mL) was added at room
temperature and stirnng continued for 2 hours. TLC (7:1:2 iPrOH:NH40H:H20)
showed
the appearance of triphosphate material. The solution was concentrated,
dissolved in water
and loaded on a DEAF Sephadex A-25 column. The column was washed with a
gradient
up to 0.6 M TEAB buffer and the product eluted off in fractions 170-179. The
fractions
were analyzed by ion exchange HPLC. Each fraction showed one triphosphate peak
that
eluted at 6.77 minutes. The fractions were combined and pumped down from
methanol
to remove buffer salt to afford 17 mg of product.
Screening for Novel Enzymatic nucleic acid molecule Motifs Using Modified NTPs
(Class
I Moti
Our initial pool contained 3 x 1014 individual sequences of 2'-amino-dCTP/2'-
amino-dUTP RNA. We optimized transcription conditions in order to increase the
amount
of RNA product by inclusion of methanol and lithium chloride. 2'-amino-2'-
deoxynucleotides do not interfere with the reverse transcription and
amplification steps of
selection and confer nuclease resistance. We designed the pool to have two
binding arms
complementary to the substrate, separated by the random 40 nucleotide region.
The 16-
mer substrate had two domains, S and 10 nucleotides long, that bind the pool,
separated by
an unpaired guanosine. On the 5' end of the substrate was a biotin attached by
a C18
linker. This enabled us to link the substrate to a NeutrAvidinTM resin in a
column format.
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The desired reaction would be cleavage at the unpaired G upon addition of
magnesium
cofactor followed by dissociation from the column due to instability of the S
base pair
helix. A detailed protocol follows:
Enzymatic nucleic acid molecule Pool Prep: The initial pool DNA was prepared
by
converting the following template oligonucleotides into double-stranded DNA by
filling in
with taq polymerise. (template=5'-ACC CTC ACT AAA GGC CGT (I~4o GGT TGC
ACA CCT TTC-3'; primer 1=S'- CAC TTA GCA TTA ACC CTC ACT AAA GGC
CGT-3'; primer 2=5'-TAA TAC GAC TCA CTA TAG GAA AGG TGT GCA ACC-3'.)
All DNA oligonucleotides were synthesized by Operon technologies. Template
oligos
were purified by denaturing PAGE and Sep-pak chromatography columns (Waters).
RNA
substrate oligos were using standard solid phase chemistry and purified by
denaturing
PAGE followed by ethanol precipitation. Substrates for in vitro cleavage
assays were 5'-
end labeled with gamma-32P-ATP and T4 polynucleotide kinase followed by
denaturing
PAGE purification and ethanol precipitation.
5 nmole of template, 10 nmole of each primer and 250 U taq polymerise were
incubated in a 10 ml volume with 1X PCR buffer (10 mM tris-HCl (pH 8.3), 1.5
mM
MgCl2, 50 mM KCl) and 0.2 mM each dNTP as follows: 94°C, 4 minutes;
(94°C, 1 min;
42°C, 1 min; 72°C, 2 min) through four cycles; and then
72°C, for 10 minutes. The
product was analyzed on 2% SeparideTM agarose gel for size and then was
extracted twice
with buffered phenol, then chloroform-isoamyl alcohol, and ethanol
precipitated. The
initial RNA pool was made by transcription of 500 pmole (3 x 10'4 molecules)
of this
DNA as follows. Template DNA was added to 40 mM tris-HCl (pH 8.0), 12 mM
MgCl2,
S mM dithiothreitol (DTT), 1 mM spermidine, 0.002% triton X-100, 1 mM LiCI, 4%
PEG-8000, 10% methanol, 2 mM ATP, 2 mM GTP, 2 mM 2'-amino-dCTP, 2 mM 2'-
amino-dUTP, 5 U/ml inorganic pyrophosphatase, and 5 U/pl T7 RNA polymerise at
room
temperature for a total volume of 1 ml. A separate reaction contained a trace
amount of
alpha-32P-GTP for detection. Transcriptions were incubated at 37°C for
2 hours followed
by addition of equal volume STOP buffer (94% formamide, ~20 mM EDTA, 0.05%
bromophenol blue). The resulting RNA was purified by 6% denaturing PAGE gel,
Sep-
pakTM chromatography, and ethanol precipitated.
INITIAL SELECTION: 2 nmole of 16 mer 5'-biotinylated substrate (S'-biotin-C18
linker-GCC GUG GGU UGC ACA C-3') was linked to 200 p1 UltraLink Immobilized
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NeutrAvidinTM resin (400 ~1 slurry, Pierce) in binding buffer (20 mM NaP04 (pH
7.5), 150
mM NaCI) for 30 minutes at room temperature. The resulting substrate column
was
washed with 2 ml binding buffer followed by 2 ml column buffer (50 mM tris-HCl
(pH
8.5), 100 mM NaCI, 50 mM KCl). The flow was capped off and 1000 pmole of
initial
pool RNA in 200 ~1 column buffer was added to the column and incubated 30
minutes at
room temperature. The column was uncapped and washed with 2 ml column buffer,
then
capped off. 200 p1 elution buffer (=column buffer + 25 mM MgCl2) was added to
the
column and allowed to incubate 30 minutes at room temperature. The column was
uncapped and eluent collected followed by three 200 u1 elution buffer washes.
The
eluent/washes were ethanol precipitated using glycogen as Garner and
rehydrated in SO ~1
sterile HzO. The eluted RNA was amplified by standard reverse
transcription/PCR
amplification techniques. 5-31 p1 RNA was incubated with 20 pmol of primer 1
in 14 w1
volume 90° for 3 min then placed on ice for 1 minute. The following
reagent were added
(final concentrations noted): 1X PCR buffer, 1 mM each dNTP, 2 U/~1 RNase
Inhibitor,
10 U/pl SuperScriptTM II reverse transcriptase. The reaction was incubated
42° for 1 hour
followed by 95° for 5 min in order to inactivate the reverse
transcriptase. The volume was
then increased to 100 ~1 by adding water and reagents for PCR: 1X PCR buffer,
20 pmol
primer 2, and 2.5 U taq DNA polymerase. The reaction was cycled in a Hybaid
thermocycler: 94°, 4 min; (94°C, 30 sec; 54°C, 30 sec;
72°C, 1 min) X 25; 72°C, 5 min.
Products were analyzed on agarose gel for size and ethanol precipitated. One-
third to one-
fifth of the PCR DNA was used to transcribe the next generation, in 100 p1
volume, as
described above. Subsequent rounds used 20 pmol RNA for the column with 40
pmol
substrate.
TWO COL UMN SELECTION: At generation 8 (G8), the column selection was
changed to the two column format. 200 pmoles of 22 mer 5'-biotinylated
substrate (S'-
biotin-C 18 linker-GCC GUG GGU UGC ACA CCU UUC C-C 18 linker-thiol modifier C6
S-S-inverted abasic-3') was used in the selection column as described above.
Elution was
in 200 p1 elution buffer followed by a 1 ml elution buffer wash. The 1200 p.1
eluent was
passed through a product trap column by gravity. The product trap column was
prepared
as follows: 200 pmol 16 mer 5'-biotinylated "product" (5'-GGU UGC ACA CCU UUC
C-C18 linker-biotin-3') was linked to the column as described above and the
column was
equilibrated in elution buffer. Eluent from the product column was
precipitated as
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previously described. The products were amplified as above only with 2.5-fold
more
volume and 100 pmol each primer. 100 ~1 of the PCR reaction was used to do a
cycle
course; the remaining fraction.was amplified the minimal number of cycles
needed for
product. After 3 rounds (G11), there was visible activity in a single turnover
cleavage
assay. By generation 13, 45% of the substrate was cleaved at 4 hours;
k°bs of the pool was
0.037 miri' in 25 mM MgCl2. We subcloned and sequenced generation 13; the pool
was
still very diverse. Since our goal was a enzymatic nucleic acid molecule that
would work
in a physiological environment, we decided to change selection pressure rather
than
exhaustively catalog G13.
Reselection of the N40 pool was started from G12 DNA. Part of the G12 DNA was
subjected to hypermutagenic PCR (Vartanian et al., 1996, Nucleic Acids
Research 24,
2627-2631) to introduce a 10% per position mutation frequency and was
designated
N40H. At round 19, part of the DNA was hypermutagenized again, giving N40M and
N40HM (a total of 4 parallel pools). The column substrates remained the same;
buffers
were changed and temperature of binding and elution was raised to 37°C.
Column buffer
was replaced by physiological buffer (50 mM tris-HCl (pH 7.5), 140 mM KCI, 10
mM
NaCI) and elution buffer was replaced by 1 mM Mg buffer (physiological buffer
+ 1 mM
MgCl2). Amount of time allowed for the pool to bind the column was eventually
reduced
to 10 min and elution time was gradually reduced from 30 min to 20 sec.
Between rounds
18 and 23, k°bs for the N40 pool stayed relatively constant at 0.035-
0.04 miri 1. Generation
22 from each of the 4 pools was cloned and sequenced.
CLONING AND SEQUENCING: Generations 13 and 22 were cloned using
Novagen's Perfectly BluntTM Cloning kit (pT7Blue-3 vector) following the kit
protocol.
Clones were screened for insert by PCR amplification using vector-specific
primers.
Positive clones were sequenced using ABI Prism 7700 sequence detection system
and
vector-specific primer. Sequences were aligned using MacVector software; two-
dimensional folding was performed using Mulfold software ( Zuker" 1989,
Science 244,
48-52; Jaeger et al., 1989, Biochemistry 86, 7706-7710; Jaeger et al., 1989,
R. F. Doolittle
ed., Methods in Enzymology, 183, 281-306). Individual clone transcription
units were
constructed by PCR amplification with SO pmol each primer 1 and primer 2 in 1X
PCR
buffer, 0.2 mM each dNTP, and 2.5 U of taq polymerase in 100 ~1 volume cycled
as
follows: 94°C, 4 min; (94°C, 30 sec; 54°C, 30 sec;
72°C, 1 min) X 20; 72°C, 5 min.
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Transcription units were ethanol precipitated, rehydrated in 30 p1 H20, and 10
~1 was
transcribed in 100 p1 volume and purified as previously described.
Thirty-six clones from each pool were sequenced and were found to be
variations of
the same consensus motif. Unique clones were assayed for activity in 1 mM
MgCl2 and
physiological conditions; nine clones represented the consensus sequence and
were used in
subsequent experiments. There were no mutations that significantly increased
activity;
most of the mutations were in regions believed to be duplex, based on the
proposed
secondary structure. In order to make the motif shorter, we deleted the 3'-
terminal 25
nucleotides necessary to bind the primer for amplification. The measured rates
of the full
length and truncated molecules were both 0.04 miri'; thus we were able reduce
the size of
the motif from 86 to 61 nucleotides. The molecule was shortened even further
by
truncating base pairs in the stem loop structures as well as the substrate
recognition arms
to yield a 48 nucleotide molecule. In addition, many of the ribonucleotides
were replaced
with 2-O-methyl modified nucleotides to stabilize the molecule. An example of
the new
motif is given in Figure 13. Those of ordinary skill in the art will recognize
that the
molecule is not limited to the chemical modifications shown in the figure and
that it
represents only one possible chemically modified molecule.
Kinetic Analysis:
Single turnover kinetics were performed with trace amounts of 5'-3zP-labeled
substrate and 10-1000 nM pool of enzymatic nucleic acid molecule. 2X substrate
in 1X
buffer and 2X pool/enzymatic nucleic acid molecule in 1X buffer were incubated
separately 90° for 3 min followed by equilibration to 37° for 3
min. Equal volume of 2X
substrate was added to pool/enzymatic nucleic acid molecule at to and the
reaction was
incubated at 37°C. Time points were quenched in 1.2 vol STOP buffer on
ice. Samples
were heated to 90°C for 3 min prior to separation on 15% sequencing
gels. Gels were
imaged using a PhosphorImager and quantitated using ImageQuantTM software
(Molecular
Dynamics). Curves were fit to double-exponential decay in most cases, although
some of
the curves required linear fits.
STABILITY: Serum stability assays were performed as previously described
(Beigelman et al., 1995, J. Biol. Chem. 270, 25702-25708). 1 wg of 5'-3zP-
labeled
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synthetic enzymatic nucleic acid molecule was added to 13 ~1 cold and assayed
for decay
in human serum. Gels and quantitation were as described in kinetics section.
SUBSTRATE REQUIREMENTS: Table 60 outlines the substrate requirements for
Class I motif. Substrates maintained Watson-Crick or wobble base pairing with
mutant
Class I constructs. Activity in single turnover kinetic assay is shown
relative to wild type
Class I and 22 mer substrate (50 mM Tris-HCL (pH 7.5), 140 mM KCI, 10 mM NaCI,
1
mM MgCl2 , 100 nM ribozyme, 5 nM substrate, 37°C).
RANDOMREGIONMUTATIONALIGNMENT.~ Table 61 outlines the random
region alignment of 134 clones from generation 22 (l .x = N40, 2.x = N40M, 3.x
= N40H,
4.x = N40HM). The number of copies of each mutant is in parenthesis in the
table,
deviations from consensus are shown. Mutations that maintain base pair U19:A34
are
shown in italic. Activity in single turnover kinetic assay is shown relative
to the G22 pool
rate (50 mM Tris-HCL pH 7.5, 140 mM KCI, 10 mM NaCI, 1 mM MgCl2 , 100 nM
ribozyme, trace substrate, 37°C).
STEM TRUNCATIONAND LOOP REPLACEMENT ANALYSIS: Figure 25 shows
a representation of Class I ribozyme stem truncation and loop replacement
analysis. The
Kre1 is compared to a 61 mer Class I ribozyme measured as described above.
Figure 26
shows examples of Class I ribozymes with truncated stems) and/or non-
nucleotide linker
replaced loop structures.
Inhibition of HCV Using Class I (Amberzyme) Motif
During HCV infection, viral RNA is present as a potential target for enzymatic
nucleic acid molecule cleavage at several processes: uncoating, translation,
RNA
replication and packaging. Target RNA may be more or less accessible to
enzymatic
nucleic acid molecule cleavage at any one of these steps. Although the
association
between the HCV initial ribosome entry site (IRES) and the translation
apparatus is
mimicked in the HCV 5'UTR/luciferase reporter system, these other viral
processes are
not represented in the OST7 system. The resulting RNA/protein complexes
associated
with the target viral RNA are also absent. Moreover, these processes may be
coupled in an
HCV-infected cell which could further impact target RNA accessibility.
Therefore, we
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tested whether enzymatic nucleic acid molecules designed to cleave the HCV
5'UTR
could effect a replicating viral system.
Recently, Lu and Wimmer characterized an HCV-poliovirus chimera in which the
poliovirus IRES was replaced by the IRES from HCV (Lu & Wimmer, 1996, Proc.
Natl.
Acad. Sci. USA. 93, 1412-1417). Poliovirus (PV) is a positive strand RNA virus
like
HCV, but unlike HCV is non-enveloped and replicates efficiently in cell
culture. The
HCV-PV chimera expresses a stable, small plaque phenotype relative to wild
type PV.
The capability of the new enzymatic nucleic acid molecule motifs to inhibit
HCV
RNA intracellularly was tested using a dual reporter system that utilizes both
firefly and
Renilla luciferase (Figure 14). A number of enzymatic nucleic acid molecules
having the
new class I motif (Amberzyme) were designed and tested (Table 56). The
Amberzyme
ribozymes were targeted to the 5' HCV UTR region, which when cleaved, would
prevent
the translation of the transcript into luciferase. OST-7 cells were plated at
12,500 cells per
well in black walled 96-well plates (Packard) in medium DMEM containing 10%
fetal
bovine serum, 1% pen/strep, and 1% L-glutamine and incubated at 37°C
overnight. A
plasmid containing T7 promoter expressing 5' HCV UTR and firefly luciferase
(T7C1-341
(Wang et al., 1993, J. of Yirol. 67, 3338-3344)) was mixed with a pRLSV40
Renilla
control plasmid (Promega Corporation) followed by enzymatic nucleic acid
molecule, and
cationic lipid to make a SX concentration of the reagents (T7C1-341 (4 pg/ml),
pRLSV40
renilla luciferase control (6 pg/ml), enzymatic nucleic acid molecule (250
nM),
transfection reagent (28.5 pg/ml).
The complex mixture was incubated at 37°C for 20 minutes. The
media was
removed from the cells and 120 p1 of Opti-mem media was added to the well
followed by
p,1 of the SX complex mixture. 1 SO p1 of Opti-mem was added to the wells
holding the
25 untreated cells. The complex mixture was incubated on OST-7 cells for 4
hours, lysed
with passive lysis buffer (Promega Corporation) and luminescent signals were
quantified
using the Dual Luciferase Assay Kit using the manufacturer's protocol (Promega
Corporation). The data shown in Figure 15 is a dose curve of enzymatic nucleic
acid
molecule targeting site 146 of the HCV RNA and is presented as a ratio between
the firefly
30 and Renilla luciferase fluorescence. The enzymatic nucleic acid molecule
was able to
reduce the quantity of HCV RNA at all enzymatic nucleic acid molecule
concentrations
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yielding an ICSO of approximately S nM. Other sites were also efficacious
(Figure 16), in
particular enzymatic nucleic acid molecules targeting sites 133, 209, and 273
were also
able to reduce HCV RNA compared to the irrelevant (1RR) controls.
Cleavage of Substrates Using Completely Modified class I (Amberzyrnel
enzymatic
nucleic acid molecule
The ability of an enzymatic nucleic acid, which is modified at every 2'
position to
cleave a target RNA was tested to determine if any ribonucleotide positions
are necessary
in the Amberzyme motif. Enzymatic nucleic acid molecules were constructed with
2'-O-
methyl, and 2'-amino (NHz) nucleotides and included no ribonucleotides (Table
56; gene
name: no ribo) and kinetic analysis was performed as described in example 13.
100 nM
enzymatic nucleic acid was mixed with trace amounts of substrate in the
presence of 1 mM
MgClz at physiological conditions (37°C). The Amberzyme with no
ribonucleotide
present in it has a Kre~ of 0.13 compared to the enzymatic nucleic acid with a
few
ribonucleotides present in the molecule shown in Table 56 (ribo). This shows
that
Amberzyme enzymatic nucleic acid molecule may not require the presence of 2'-
OH
groups within the molecule for activity.
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Substrate Recognition Rules for Class II (zinzyme) enzymatic nucleic acid
molecules
Class II (zinzyme) ribozymes were tested for their ability to cleave base-
paired
substrates with all sixteen possible combinations of bases immediately 5' and
3' proximal
to the bulged cleavage site G. Ribozymes were identical in all remaining
positions of their
7 base pair binding arms. Activity was assessed at two and twenty-four hour
time points
under standard reaction conditions [20 mM HEPES pH 7.4, 140 mM KCI, 10 mM
NaCI, 1
mM MgCl2, 1 mM CaCl2 - 37° C]. Figure 19 shows the results of this
study. Base paired
substrate UGG (not shown in the figure) cleaved as poorly as CGG shown in the
figure.
The figure shows the cleavage site substrate triplet in the 5'- 3' direction
and 2 and 24
hour time points are shown top to bottom respectively. The results indicate
the cleavage
site triplet is most active with a 5'- Y-G-H -3' (where Y is C or U and H is
A, C or U with
cleavage between G and H); however, activity is detected particularly with the
24 hour
time point for most paired substrates. All positions outside of the cleavage
triplet were
found to tolerate any base pairings (data not shown).
All possible mispairs immediately 5' and 3' proximal to the bulged cleavage
site G
were tested to a class II ribozyme designed to cleave a 5'-C-G-C -3'. It was
observed the
5' and 3' proximal sites are as active with G:U wobble pairs, in addition, the
5' proximal
site will tolerate a mismatch with only a slight reduction in activity [data
not shown].
Screening for Novel Enzymatic nucleic acid molecule Motifs (Class II Motifs)
The selections were initiated with pools of > 1014 modified RNA's of the
following
sequence: 5'-GGGAGGAGGAAGUGCCU (N)35 UGCCGCGCUCGCUCCCAGUCC-3'. The RNA
was enzymatically generated using the mutant T7 Y639F RNA polymerise prepared
by
Rui Souza. The following modified NTP's were incorporated: 2'-deoxy-2'-fluoro-
adenine triphosphate, 2'-deoxy-2'-fluoro-uridine triphosphate or 2'-deoxy-2'-
fluoro-5-[(N-
imidazole-4acetyl)propyl amine] uridine triphosphate, and 2'-deoxy-2'-amino-
cytidine
triphosphate; natural guanidine triphosphate was used in all selections so
that alpha 32P-
GTP could be used to label pool RNA's. RNA pools were purified by denaturing
gel
electrophoresus 8% polyacrilamide 7 M Urea.
The following target RNA (resin A) was synthesized and coupled to Iodoacetyl
UltralinkTM resin (Pierce) by the supplier's proceedurea' -b-L-
GGACUGGGAGCGAGCGCGGCGCAGGCACU GAAG-L-S-B-3'; where b is biotin (Glenn
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Research cat# 10-1953-nn), L is polyethylene glycol spacer (Glenn Research
cat# 10-1918-
nn), S is thiol-modifier C6 S-S (Glenn Research cat# 10-1936-nn), B is a
standard inverted
deoxy abasic.
RNA pools were added to 100 ~l of 5 uM Resin A in the buffer A (20 mM HEPES
pH 7.4, 140 mM KCL, 10 mM NaCI) and incubated at 22°C for 5 minutes.
The
temperature was then raised to 37°C for 10 minutes. The resin was
washed with 5 ml
buffer A. Reaction was triggered by the addition of buffer B(20 mM HEPES pH
7.4, 140
mM KCL, 10 mM NaCI, 1 mM MgCl2, 1 mM CaCl2). Incubation proceeded for 20
minutes in the first generation and was reduced progressively to 1 minute in
the final
generations; with 13 total generations. The reaction eluent was collected in 5
M NaCI to
give a final concentration of 2 M NaCI. To this was added 100 p,1 of 50%
slurry Ultralink
NeutraAvidinTM (Pierce). Binding of cleaved biotin product to the avidin resin
was
allowed by 20 minute incubation at 22° C. The resin was subsequently
washed with 5 ml
of 20 mM HEPES pH 7.4, 2 M NaCI. Desired RNA's were removed by a 1.2 ml
denaturing wash 1M NaCI, 10 M Urea at 94° C over 10 minutes. RNA's were
double
precipitated in 0.3 M sodium acetate to remove Cl- ions inhibitory to reverse
transcription.
Standard protocols of reverse transcription and PCR amplification were
performed.
RNA's were again transcribed with the modified NTP's described above. After 13
generations cloning and sequencing provided 14 sequences which were able to
cleave the
target substrate. Six sequences were characterized to determine secondary
structure and
kinetic cleavage rates. The structures and kinetic data are given in Figure
17. The
sequences of eight other enzymatic nucleic acid molecule sequences are given
in Table 57.
The size, sequence, and chemical compositions of these molecules can be
modified as
described below or using other techniques well known in the art.
Nucleic Acid Catalyst En~,ineerin~
Sequence, chemical and structural variants of Class I and Class II enzymatic
nucleic
acid molecule can be engineered and re-engineered using the techniques shown
in this
application and known in the art. For example, the size of class I and class
II enzymatic
nucleic acid molecules can, be reduced or increased using the techniques known
in the art
(Zaug et al., 1986, Nature, 324, 429; Ruffner et al., 1990, Biochem., 29,
10695; Beaudry et
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al., 1990, Biochem., 29, 6534; McCall et al., 1992, Proc. Natl. Acad. Sci.,
USA., 89, 5710;
Long et al., 1994, supra; Hendry et al., 1994, BBA 1219, 405; Benseler et al.,
1993, JACS,
115, 8483; Thompson et al., 1996, Nucl. Acids Res., 24, 4401; Michels et al.,
1995,
Biochem., 34, 2965; Been et al., 1992, Biochem., 31, 11843; Guo et al., 1995,
EMBO. J.,
14, 368; Pan et al., 1994, Biochem., 33, 9561; Cech, 1992, Curr. Op. Struc.
Bio., 2, 605;
Sugiyama et al., 1996, FEBS Lett., 392, 215; Beigelman et al., 1994, Bioorg.
Med. Chem.,
4, 171 S; Santoro et al., 1997, PNAS 94, 4262; all are incorporated in their
totality by
reference herein), to the extent that the overall catalytic activity of the
ribozyme is not
significantly decreased.
Further rounds of in vitro selection strategies described herein and
variations thereof
can be readily used by a person skilled in the art to evolve additional
nucleic acid catalysts
and such new catalysts are within the scope of the instant invention.
Example 16' Activit~r of Class II (zinzyme) nucleic acid catalysts to inhibit
HER2 gene
expression
Applicant has designed, synthesized and tested several class II (zinzyme)
ribozymes
targeted against HER2 RNA (see, for example, Tables 58, 59, and 62) in cell
proliferation
RNA reduction assays.
Proliferation assay:
The model proliferation assay used in the study can require a cell-plating
density of
2000-10000 cells/well in 96-well plates and at least 2 cell doublings over a 5-
day treatment
period. Cells used in proliferation studies were either human breast or
ovarian cancer cells
(SKBR-3 and SKOV-3 cells respectively). To calculate cell density for
proliferation
assays, the FIPS (fluoro-imaging processing system) method well known in the
art was
used. This method allows for cell density measurements after nucleic acids are
stained
with CyQuant~ dye, and has the advantage of accurately measuring cell
densities over a
very wide range 1,000-100,000 cells/well in 96-well format.
Ribozymes (50-200 nM) were delivered in the presence of cationic lipid at 2.0-
5.0
pg/mL and inhibition of proliferation was determined on day S post-treatment.
Two full
ribozyme screens were completed resulting in the selection of 14 ribozymes.
Class II
(zinzyme) ribozymes against sites, 314 (RPI No. 18653), 443 (RPI No. 18680),
597 (RPI
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No. 18697), 659 (RPI No. 18682), 878 (RPI Nos. 18683 and 18654), 881 (RPI Nos.
18684
and 18685) 934 (RPI No. 18651), 972 (RPI No. 18656, 19292, 19727, 19728, and
19293),
1292 (RPI No. 18726), 1541 (RPI No. 18687), 2116 (RPI No. 18729), 2932 (RPI
No.
18678), 2540 (RPI No. 18715), and 3504 (RPI No. 18710) caused inhibition of
proliferation ranging from 25-80% as compared to a scrambled control ribozyme.
An
example of results from a cell culture assay is shown in Figure 20. Refernng
to Figure
20, Class II ribozymes targeted against HER2 RNA are shown to cause
significant
inhibition of proliferation of cells. This shows that ribozymes, for instance
the Class II
(zinzyme) ribozyrnes are capable of inhibiting HER2 gene expression in
mammalian cells.
RNA assay:
RNA was harvested 24 hours post-treatment using the Qiagen RNeasy~ 96
procedure. Real time RT-PCR (TaqMan~ assay) was performed on purified RNA
samples using separate primer/probe sets specific for either target HER2 RNA
or control
actin RNA (to normalize for differences due to cell plating or sample
recovery). Results
are shown as the average of triplicate determinations of HER2 to actin RNA
levels post-
treatment. Figure 30 shows class II ribozyme (zinzyme) mediated reduction in
HER2
RNA targeting site 972 vs a scrambled attenuated control.
Dose response assays:
Active ribozyme was mixed with binding arm-attenuated control (BAC) ribozyme
to
a final oligonucleotide concentration of either 100, 200 or 400 nM and
delivered to cells in
the presence of cationic lipid at 5.0 ~tg/mL. Mixing active and BAC in this
manner
maintains the lipid to ribozyme charge ratio throughout the dose response
curve. HER2
RNA reduction was measured 24 hours post-treatment and inhibition of
proliferation was
determined on day S post-treatment. The dose response antiproliferation
results are
summarized in Figure 31 and the dose-dependent reduction of HER2 RNA results
are
summarized in Figure 32. Figure 33 shows a combined dose response plot of both
anti-
proliferation and RNA reduction data for a class II ribozyme targeting site
972 of HER2
RNA (RPI 19293).
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Example 17' Reduction of ribose residues in Class II (zinzyme) nucleic acid
catalysts
Class II (zinzyme) nucleic acid catalysts were tested for their activity as a
function
ribonucleotide content. A Zinzyme having no ribonucleotide residue (ie., no 2'-
OH group
at the 2' position of the nucleotide sugar) against the K-Ras site 521 was
designed. This
molecules were tested utilizing the chemistry shown in Figure 27a. The in
vitro catalytic
activity zinzyme construct was not significantly effected (the cleavage rate
reduced only
fold).
The Kras zinzyme shown in Figure 27a was tested in physiological buffer with
the
divalent concentrations as indicated in the legend (high NaCI is an altered
monovalent
10 condition shown) of Figure 28. The 1 mM Cap condition yielded a rate of
0.005 miri'
while the 1 mM Mgr condition yielded a rate of 0.002 miri 1. The ribose
containing wild
type yields a rate of 0.05 miri' while substrate in the absence of zinzyme
demonstrates less
than 2% degradation at the longest time point under reaction conditions shown.
This
illustrates a well-behaved cleavage reaction done by a non-ribose containing
catalyst with
only a 10-fold reduced cleavage as compared to ribonucleotide-containing
zinzyme and
vastly above non-catalyzed degradation.
A more detailed investigation into the role of ribose positions in the Class
II
(zinzyme) motif was carried out in the context of the HER2 site 972 (Applicant
has further
designed a fully modified Zinzyme as shown in Figure 27b targeting the HER2
RNA site
972). Figure 29 is a diagram of the alternate formats tested and their
relative rates of
catalysis. The effect of substitution of ribose G for the 2'-O-methyl C-2'-O-
methyl A in
the loop of Zinzyme (see Figure 34) was insignificant when assayed with the
Kras target
but showed a modest rate enhancement in the HER2 assays. The activity of all
Zinzyme
motifs, including the fully stabilized "0 ribose" (RPI 19727) are well above
background
noise level degradation. Zinzyme with only two ribose positions (RPI 19293)
are
sufficient to restore "wild-type" activity. Motifs containing 3 (RPI 19729), 4
(RPI 19730)
or 5 ribose (RPI 19731) positions demonstrated a greater extent of cleavage
and profiles
almost identical to the 2 ribose motif. Applicant has thus demonstrated that a
Zinzyme
with no ribonucleotides present at any position can catalyze efficient RNA
cleavage
activity. Thus, Zinzyme enzymatic nucleic acid molecules do not require the
presence of
2'-OH group within the molecule for catalytic activity.
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Example 18: Activity of reduced ribose containin Class II zinzyme) nucleic
acid
cata~sts to inhibit HER2 gene expression
A cell proliferation assay-for testing reduced ribo class II (zinzyme) nucleic
acid
catalysts (50-400 nM) targeting HER2 site 972 was performed as described in
example 19.
The results of this study are summarized in Figure 35. These results indicate
significant
inhibition of HER2 gene expression using stabilized Class II (zinzyme) motifs,
including
two ribo (RPI 19293), one ribo (RPI 19728), and non-ribo (RPI 19727)
containing nucleic
acid catalysts.
Applications
The use of NTP's described in this invention have several research and
commercial
applications. These modified nucleotide triphosphates can be used for in vitro
selection
(evolution) of oligonucleotides with novel functions. Examples of in vitro
selection
protocols are incorporated herein by reference (Joyce, 1989, Gene, 82, 83-87;
Beaudry et
al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97;
Breaker et
al., 1994, TIBTECH 12, 268; Bartel et al.,1993, Science 261:1411-1418;
Szostak, 1993,
TIBS 17, 89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr.
Op. Biotech.,
7, 442).
Additionally, these modified nucleotide triphosphates can be employed to
generate
modified oligonucleotide combinatorial chemistry libraries. Several references
for this
technology exist (Brenner et al., 1992, PNAS 89, 5381-5383, Eaton, 1997, Curr.
Opin.
Chem. Biol. 1, 10-16), which are all incorporated herein by reference.
Diagnostic uses
Enzymatic nucleic acid molecules of this invention may be used as diagnostic
tools
to examine genetic drift and mutations within diseased cells or to detect the
presence of
specific RNA in a cell. The close relationship between enzymatic nucleic acid
molecule
activity and the structure of the target RNA allows the detection of mutations
in any region
of the molecule which alters the base-pairing and three-dimensional structure
of the target
RNA. By using multiple enzymatic nucleic acid molecules described in this
invention,
one may map nucleotide changes which are important to RNA structure and
function in
vitro, as well as in cells and tissues. Cleavage of target RNAs with enzymatic
nucleic acid
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molecules may be used to inhibit gene expression and define the role
(essentially) of
specified gene products in the progression of disease. In this manner, other
genetic targets
may be defined as important mediators of the disease. These experiments will
lead to
better treatment of the disease progression by affording the possibility of
combinational
therapies (e.g., multiple enzymatic nucleic acid molecules targeted to
different genes,
enzymatic nucleic acid molecules coupled with known small molecule inhibitors,
radiation
or intermittent treatment with combinations of enzymatic nucleic acid
molecules and/or
other chemical or biological molecules). Other in vitro uses of enzymatic
nucleic acid
molecules of this invention are well known in the art, and include detection
of the presence
of mRNAs associated with related conditions. Such RNA is detected by
determining the
presence of a cleavage product after treatment with a enzymatic nucleic acid
molecule
using standard methodology.
In a specific example, enzymatic nucleic acid molecules which can cleave only
wild
type or mutant forms of the target RNA are used for the assay. The first
enzymatic nucleic
acid molecule is used to identify wild-type RNA present in the sample and the
second
enzymatic nucleic acid molecule will be used to identify mutant RNA in the
sample. As
reaction controls, synthetic substrates of both wild-type and mutant RNA will
be cleaved
by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic
nucleic
acid molecule efficiencies in the reactions and the absence of cleavage of the
"non-
targeted" RNA species. The cleavage products from the synthetic substrates
will also
serve to generate size markers for the analysis of wild type and mutant RNAs
in the
sample population. Thus each analysis can involve two enzymatic nucleic acid
molecules,
two substrates and one unknown sample which can be combined into six
reactions. The
presence of cleavage products can be determined using an RNAse protection
assay so that
full-length and cleavage fragments of each RNA can be analyzed in one lane of
a
polyacrylamide gel. It is not absolutely required to quantify the results to
gain insight into
the expression of mutant RNAs and putative risk of the desired phenotypic
changes in
target cells. The expression of mRNA whose protein product is implicated in
the
development of the phenotype is adequate to establish risk. If probes of
comparable
specific activity are used for both transcripts, then a qualitative comparison
of RNA levels
will be adequate and will decrease the cost of the initial diagnosis. Higher
mutant form to
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wild-type ratios will be correlated with higher risk whether RNA levels are
compared
qualitatively or quantitatively.
Additional Uses
Potential usefulness of sequence-specific enzymatic nucleic acid molecules of
the
instant invention can have many of the same applications for the study of RNA
that DNA
restriction endonucleases have for the study of DNA (Nathans et al., 1975 Ann.
Rev.
Biochem. 44:273). For example, the pattern of restriction fragments can be
used to
establish sequence relationships between two related RNAs, and large RNAs
could be
specifically cleaved to fragments of a size more useful for study. The ability
to engineer
sequence specificity of the enzymatic nucleic acid molecule is ideal for
cleavage of RNAs
of unknown sequence. Applicant describes the use of nucleic acid molecules to
down-
regulate gene expression of target genes in bacterial, microbial, fungal,
viral, and
eukaryotic systems including plant, or mammalian cells.
All patents and publications mentioned in the specification are indicative of
the
levels of skill of those skilled in the art to which the invention pertains.
All references
cited in this disclosure are incorporated by reference to the same extent as
if each reference
had been incorporated by reference in its entirety individually.
One skilled in the art would readily appreciate that the present invention is
well
adapted to carry out the objects and obtain the ends and advantages mentioned,
as well as
those inherent therein. The methods and compositions described herein as
presently
representative of preferred embodiments are exemplary and are not intended as
limitations
on the scope of the invention. Changes therein and other uses will occur to
those skilled in
the art, which are encompassed within the spirit of the invention, are defined
by the scope
of the claims.
It will be readily apparent to one skilled in the art that varying
substitutions and
modifications may be made to the invention disclosed herein without departing
from the
scope and spirit of the invention. Thus, such additional embodiments are
within the scope
of the present invention and the following claims.
The invention illustratively described herein suitably may be practiced in the
absence
of any element or elements, limitation or limitations which is not
specifically disclosed
herein. Thus, for example, in each instance herein any of the terms
"comprising",
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"consisting essentially of and "consisting of may be replaced with either of
the other two
terms. The terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that in the use
of such terms and
expressions of excluding any equivalents of the features shown and described
or portions
thereof, but it is recognized that various modifications are possible within
the scope of the
invention claimed. Thus, it should be understood that although the present
invention has
been specifically disclosed by preferred embodiments, optional features,
modification and
variation of the concepts herein disclosed may be resorted to by those skilled
in the art, and
that such modifications and variations are considered to be within the scope
of this
invention as defined by the description and the appended claims.
In addition, where features or aspects of the invention are described in terms
of
Markush groups or other grouping of alternatives, those skilled in the art
will recognize
that the invention is also thereby described in terms of any individual member
or subgroup
of members of the Markush group or other group.
Thus, additional embodiments are within the scope of the invention and within
the
following claims
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Table 1
TABLE 1
Characteristics of naturally occurring ribozymes
Group I Introns
~ Size: 150 to >1000 nucleotides.
~ Requires a U in the target sequence immediately 5' of the cleavage site.
~ Binds 4-6 nucleotides at the 5'-side of the cleavage site.
~ Reaction mechanism: attack by the 3'-OH of guanosine to generate cleavage
products
with 3'-OH and 5'-guanosine.
~ Additional protein cofactors required in some cases to help folding and
maintainance of
the active structure.
~ Over 300 known members of this class. Found as an intervening sequence in
Tetrah~rnena
thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-green
algae,' and
others.
~ Major structural features largely established through phylogenetic
comparisons,
mutagenesis, and biochemical studies (',i'].
~ Complete kinetic framework established for one ribozyme ("
;'°,",°'].
~ Studies of ribozyme folding and substrate docking underway ("i',""','XJ.
~ Chemical modification investigation of important residues well established
(x,Xy_
~ The small (4-6 nt) binding site may make this ribozyme too non-specific for
targeted RNA
cleavage, however, the Tetrahymena group I intron has been used to repair a
"defective'
[3-galactosidase message by the ligation of new (3-galactosidase sequences
onto the
defective message (X"].
RNAse P RNA (M1 RNA)
~ Size: 290 to 400 nucleotides.
~ RNA portion of a ubYquitous ribonucleoprotein enzyme.
~ Cleaves tRNA precursors to form mature tRNA (X"'].
~ Reaction mechanism: possible attack by MZ+-OH to generate cleavage products
with 3'-
OH and 5'-phosphate.
~ RNAse P is found throughout the prokaryotes and eukaryotes. The RNA subunit
has
been sequenced from bacteria, yeast, rodents, and primates.
~ Recruitment of endogenous RNAse P for therapeutic applications is possible
through
hybridization of an External Guide Sequence (EGS) to the target RNA
(Xi°,x°]
~ Important phosphate and 2' OH contacts recently identified (X°'~X""]
Group If Introns
Size: >1000 nucleotides.
Trans cleavage of target RNAs recently demonstrated (X""i,XiX]
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Table 1
~ Sequence requirements not fully determined.
~ Reaction mechanism: 2'-OH of an internal adenosine generates cleavage
products with 3'-
OH and a "lariat" RNA containing a 3'-5' and a 2'-5' branch point.
~ Only natural ribozyme with demonstrated participation in DNA cleavage
(XX~xx'] in
addition to RNA cleavage and ligation.
~ Major structural features largely established through phylogenetic
comparisons (xXllJ.
~ Important 2' OH contacts beginning to be identified (Xx"'~
~ Kinetic framework under development (xxi"]
Neurospora VS RNA
~ Size: ~144 nucleotides.
Trans cleavage of hairpin target RNAs recently demonstrated (XX°].
~ Sequence requirements not fully determined.
~ Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate
cleavage products
with 2';3'-cyclic phosphate and 5'-OH ends.
~ Binding sites and structural requirements not fully determined.
~ Oi-tly 1 known member of this class. Found in Neurospora VS RNA.
Hammerhead Ribozyme
(see text for references)
~ Size: ~13 to 40 nucleotides.
~ Requires the target sequence UH immediately 5' of the cleavage site.
~ Binds a variable number nucleotides on both sides of the cleavage site.
~ Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate
cleavage products
with 2',3'-cyclic phosphate and 5'-OH ends.
~ 14 known members of this class. Found in a number of plant pathogens
(virusoids) that
use RNA as the infectious agent.
~ Essential structural features largely defined, including 2 crystal
structures (xX°i~XX°ll~
~ Minimal ligation activity demonstrated (for engineering through in vitro
selection) (XX°"']
~ Complete kinetic framework established for two or more ribozymes (XX'XJ.
~ Chemical modification investigation of important residues well established
(XXX].
Hairpin Ribozyme
~ Size: ~50 nucleotides.
~ Requires the target sequence GUC immediately 3' of the cleavage site.
~ Binds 4-6 nucleotides at the 5'-side of the cleavage site and a variable
number to the 3'-
side of the cleavage site.
~ Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate
cleavage products
with 2',3'-cyclic phosphate and 5'-OH ends.
~ 3 known members of this class. Found in three plant pathogen (satellite RNAs
of the
tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus)
which uses
RNA as the infectious agent.
~ Essential structural features largely defined (XXX' XXXu XXXiii
XxX'°~
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175
Table 1
Ligation activity (in addition to cleavage activity) makes ribozyme amenable
to
engineering through in vitro selection (XXX°)
Complete kinetic framework established for one ribozyme (xXXVt].
Chemical modification investigation of important residues begun
(xXx°° xXX"iii~.
Hepatitis Delta Virus (HDV) Ribozyme
~ Size: ~60 nucleotides.
~ Trans cleavage of target RNAs demonstrated (XXXiX].
~ Binding sites and structural requirements not fully determined, although no
sequences 5'
of cleavage site are required. Folded ribozyme contains a pseudoknot structure
[x1].
~ Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate
cleavage products
with 2',3'-cyclic phosphate and 5'-OH ends.
~ Only 2 known members of this class. Found in human HDV.
~ Circular form of HDV is active and shows increased nuclease stability (Xy
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Tetrahymena thermophila
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Biochemistry (1990), 29(44), 10159-71.
Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the
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forms a mismatch at the active
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Knitt, Deborah S.; Herschlag, Daniel. pH Dependencies of the Tetrahymena
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Unconventional Origin of an Apparent pKa. Biochemistry (1996), 35(5), 1560-70.
°i . Bevilacqua, Philip C.; Sugimoto, Naoki; Turner, Douglas H.. A
mechanistic framework for the
second step of splicing catalyzed by the Tetrahymena ribozyme. Biochemistry
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w . Li, Yi; Bevilacqua, Philip C.; Mathews, David; Turner, Douglas H..
Thermodynamic and
activation parameters for binding of a pyrene-labeled substrate by the
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not diffusion-controlled and is driven by a favorable entropy change.
Biochemistry (1995), 34(44),14394-9.
Banerjee, Aloke Raj; Turner, Douglas H.. The time dependence of chemical
modification reveals
slow steps in the folding of a group I ribozyme. Biochemistry (1995), 34(19),
6504-12.
Zarrinkar, Patrick P.; Williamson, James R.. The P9.1-P9.2 peripheral
extension helps guide
folding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996), 24(5), 854-8.
X . Strobel, Scott A.; Cech, Thomas R.. Minor groove recognition of the
conserved G.cntdot.U pair at
the Tetrahymena ribozyme reaction site. Science (Washington, D. C.) (1995),
267(5198), 675-9.
Strobel, Scott A.; Cech, Thomas R.. Exocyclic Amine of the Conserved
G.cntdot.U Pair at the
Cleavage Site of the Tetrahymena Ribozyme Contributes to 5'-Splice Site
Selection and Transition State
Stabilization. Biochemistry (1996), 35(4),1201-11.
Sullenger, Bruce A.; Cech, Thomas R.. Ribozyme-mediated repair of defective
mRNA by targeted
trans-splicing. Nature (London) (1994), 371(6498), 619-22.
Robertson, H.D.; Altman, S.; Smith, J.D. J. Biol. Chem., 247, 5243-5251
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Forster, Anthony C.; Altman, Sidney. External guide sequences for an RNA
enzyme. Science
(Washington, D. C.,1883-) (1990), 249(4970), 783-6.
Yuan, Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by human RNase P.
Proc. Natl.
Acad. Sci. USA (1992) 89, 8006-10.
Harris, Michael E.; Pace, Norman R.. Identification of phosphates involved in
catalysis by the
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Table 1
ribozyme RNase P RNA. RNA (1995),1(2), 210-18.
X°u . Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary
interactions in RNA: 2'-hydroxyl-base
contacts between the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U. S. A.
(1995), 92(26),12510-14.
x..su . Pyle, Anna Marie; Green, Justin B.. Building a Kinetic Framework for
Group II Intron Ribozyme
Activity: Quantitation of Interdomain Binding and Reaction Rate. Biochemistry
(1994), 33(9), 2716-25.
Michels, William J. Jr.; Pyle, Anna Marie. Conversion of a Group II Intron
into a New Multiple-
Turnover Ribozyme that Selectively Cleaves Oligonucleotides: Elucidation of
Reaction Mechanism and .
Structure/Function Relationships. Biochemistry (1995), 34(9), 2965-77.
XX . Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang, Jian; Penman, Philip
S.; Lambowitz, Alan
M.. A group II intron RNA is a catalytic component of a DNA endonuclease
involved in intron mobility.
Cell (Cambridge, Mass.) (1995), 83(4), 529-38.
xXi . Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams J., Jr.; Pyle,
Anna Marie. Group II intron
ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack
contacts with substrate 2'-
hydroxyl groups. Chem. Biol. (1995), 2(11), 761-70.
Michel, Francois; Ferat, Jean Luc. Structure and activities of group II
introns. Annu. Rev.
Biochem. (1995), 64, 435-61.
Abramovitz, Dana L.; Friedman, Richard A.; Pyle, Anna Marie. Catalytic role of
2'-hydroxyl
groups within a group II intron active site. Science (Washington, D. C.)
(1996), 271(5254), 1410-13.
Daniels, Danette L.; Michels, William J., Jr.; Pyle, Anna Marie. Two competing
pathways for self-
splicing by group II introns: a quantitative analysis of in vitro reaction
rates and products. J. Mol. Biol.
(1996), 256(1), 31-49.
xxv . Guo, Hans C. T.; Collins, Richard A.. Efficient trans-cleavage of a stem-
loop RNA substrate by a
ribozyme derived from Neurospora VS RNA. EMBO J. (1995), 14(2), 368-76.
xX°. . Scott, W.G., Finch, J.T., Aaron,K. The crystal structure of an
all RNA hammerhead
ribozyme:Aproposed mechanism for RNA catalytic cleavage. Cell, (1995), 81, 991-
1002.
XX..u . McKay, Structure and function of the hammerhead ribozyme: an
unfinished story. RNA, (1996),
2, 395-403.
Long, D., Uhlenbeck; O., Hertel, K. Ligation with hammerhead ribozymes. US
Patent No.
5,633,133.
xXiX . Hertel, K.J., Herschlag, D., Uhlenbeck, O. A kinetic and thermodynamic
framework for the
hammerhead ribozyme reaction. Biochemistry, (1994) 33, 3374-3385.Beigelman,
L., et al., Chemical
modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-
25708.
xXX _ Beigelman, L., et al., Chemical modifications of hammerhead ribozymes.
J. Biol. Chem., (1995)
270, 25702-25708.
XXXi . Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip.
'Hairpin' catalytic RNA model:
evidence for helixes and sequence requirement for substrate RNA. Nucleic Acids
Res. (1990), 18(2), 299-
304.
xxXia . Chowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke, John M.. Novel
guanosine requirement for
catalysis by the hairpin ribozyme. Nature (London) (1991), 354(6351), 320-2.
Berzal-Herranz, Alfredo; Joseph, Simpson; Chowrira, Bharat M.; Butcher, Samuel
E.; Burke, John
M.. Essential nucleotide sequences and secondary structure elements of the
hairpin ribozyme. EMBO J.
(1993),12(6), 2567-73.
XXXi° . Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat M.;
Butcher, Samuel E.. Substrate
selection rules for the hairpin ribozyme determined by in vitro selection,
mutation, and analysis of
mismatched substrates. Genes Dev. (1993), 7(1),130-8.
XxX° . Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John M.. In
vitro selection of active hairpin
ribozymes by sequential RNA-catalyzed cleavage and ligation reactions. Genes
Dev. (1992), 6(1), 129-34.
xXX°i . Hegg, Lisa A.; Fedor, Martha J.. Kinetics and Thermodynamics of
Intermolecular Catalysis by
Hairpin Ribozymes. Biochemistry (1995), 34(48),15813-28.
xxxvii . Grasby, Jane A.; Mersmann, Karin; Singh, Mohinder; Gait, Michael J..
Purine Functional Groups
in Essential Residues of the Hairpin Ribozyme Required for Catalytic Cleavage
of RNA. Biochemistry
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Table 1
(1995), 34(12), 4068-76. . .
xXx°v~ . Schmidt, Sabine; Beigelman, Leonid; Karpeisky, Alexander;
Usman, Nassim; Sorensen, Ulrik S.;
Gait, Michael J.. Base and sugar requirements for RNA cleavage of essential
nucleoside residues in
internal loop B of the hairpin ribozyme: implications for secondary structure.
Nucleic Acids Res. (1996),
24(4), 573-81.
xXXcX . Perrotta, Anne T.; Been, Michael D.. Cleavage of oligoribonucleotides
by a ribozyme derived from
the hepatitis .delta. virus RNA sequence. Biochemistry (1992), 31(1),16-21.
Perrotta, Anne T.; Been, Michael D.. A pseudoknot-like structure required for
efficient self-
cleavage of hepatitis delta virus RNA. Nature (London) (1991), 350(6317), 434-
6.
xli . Puttaraju, M.; Perrotta, Anne T.; Been, Michael D.. A circular trans-
acting hepatitis delta virus
ribozyme. Nucleic Acids Res. (1993), 21(18), 4253-8.
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180
Table 3
Table 3: Human PTP-1B Hammerhead Ribozyme and Target Sequence
Nt. Riboryme Seq. Substrate Seq.
Position Sequence ID Sequence ID
Nos. Nos.
15 UGCCGCUCCUGAUGAGX CGAA AGGCCGCG1 CGCGGCCTA GAGCGGCA529
72 AUCUCCAUCUGAUGAGX CGAA ACGGGCCA2 ' TGGCCCGTC ATGGAGAT530
92 UCUGCUCGCUGAUGAGX CGAA ACUCCUW3 AAAGGAGTT CGAGCAGA531
93 AUCUGCUCCUGAUGAGX CGAA AACUCCUU4 AAGGAGTTC GAGCAGAT532
102 GACWGUC CUGAUGAGX CGAA AUCUGCUC5 GAGCAGATC GACAAGTC533
110 AGCUCCCGCUGAUGAGX CGAA ACWGUCG6 CGACAAGTC CGGGAGCT534
129 UCCUGGUACUGAUGAGX CGAA AUGGCCGC7 GCGGCCATT TACCAGGA535
130 AUCCUGGUCUGAUGAGX CGAA AAUGGCCG8 CGGCCATTT ACCAGGAT536
131 UAUCCUGGCUGAUGAGX CGAA AAAUGGCC9 GGCCATTTA CCAGGATA537
139 AUGUCGGACUGAUGAGX CGAA AUCCUGGU10 ACCAGGATA TCCGACAT538
141 UCAUGUCGCUGAUGAGX CGAA AUAUCCUG11 CAGGATATC CGACATGA539
161 UACAUGGGCUGAUGAGX CGAA AGUCACUG12 CAGTGACTT CCCATGTA540
162 CUACAUGGCUGAUGAGX CGAA AAGUCACU13 AGTGACTTC CCATGTAG541
169 GGCCACUCCUGAUGAGX CGAA ACAUGGGA14 TCCCATGTA GAGTGGCC542
183 WCWAGG CUGAUGAGX CGAA AGCWGGC15 GCCAAGCTT CCTAAGAA543
184 GWCWAG CUGAUGAGX CGAA AAGCWGG16 CCAAGCTTC CTAAGAAC544
187 UUUGUUCU X CGAA AGGAAGCU17 AGCTTCCTA AGAACAAA545
CUGAUGAG
20S UCUGUACCCUGAUGAGX CGAA AUWCGGU18 ACCGAAATA GGTACAGA546
209 CGUCUCUGCUGAUGAGX CGAA ACCUAUW19 AAATAGGTA CAGAGACG547
219 AAGGGACUCUGAUGAGX CGAA ACGUCUCU20 AGAGACGTC AGTCCCTT548
223 GUCAAAGGCUGAUGAGX CGAA ACUGACGU21 ACGTCAGTC CCTTTGAC549
227 UAUGGUCACUGAUGAGX CGAA AGGGACUG22 CAGTCCCTT TGACCATA550
228 CUAUGGUCCUGAUGAGX CGAA AAGGGACU23 AGTCCCTTT GACCATAG551
235 AAUCCGACCUGAUGAGX CGAA AUGGUCAA24 TTGACCATA GTCGGATT552
238 UUUAAUCCCUGAUGAGX CGAA ACUAUGGU25 ACCATAGTC GGATTAAA553
243 UGUAGUW X CGAA AUCCGACU26 AGTCGGATT AAACTACA554
CUGAUGAG
244 AUGUAGUUCUGAUGAGX CGAA AAUCCGAC27 GTCGGATTA AACTACAT555
249 UCWGAUG CUGAUGAGX CGAA AGUWAAU28 ATTAAACTA CATCAAGA556
253 AUCUUCUU X CGAA AUGUAGW29 AACTACATC AAGAAGAT557
CUGAUGAG
262 AUAGUCAUCUGAUGAGX CGAA AUCWCUU30 AAGAAGATA ATGACTAT558
269 CGWGAUA CUGAUGAGX CGAA AGUCAWA31 TAATGACTA TATCAACG559
271 AGCGWGA CUGAUGAGX CGAA AUAGUCAU32 ATGACTATA TCAACGCT560
273 CUAGCGUUCUGAUGAGX CGAA AUAUAGUC33 GACTATATC AACGCTAG561
280 UAUCAAACCUGAUGAGX CGAA AGCGWGA34 TCAACGCTA GTTTGATA562
283 UUUUAUCACUGAUGAGX CGAA ACUAGCGU35 ACGCTAGTT TGATAAAA563
284 UUUWAUC CUGAUGAGX CGAA AACUAGCG36 CGCTAGTTT GATAAAAA564
288 UCCAUUW CUGAUGAGX CGAA AUCAAACU37 AGTTTGATA AAAATGGA565
313 AAGAAUGUCUGAUGAGX CGAA ACUCCUW38 AAAGGAGTT ACATTCTT566
314 UAAGAAUGCUGAUGAGX CGAA AACUCCUU39 AAGGAGTTA CATTCTTA567
318 UGGGUAAGCUGAUGAGX CGAA AUGUAACU40 AGTTACATT CTTACCCA568
319 CUGGGUAA X CGAA AAUGUAAC41 GTTACATTC TTACCCAG569
CUGAUGAG
321 CCCUGGGUCUGAUGAGX CGAA AGAAUGUA42 TACATTCTT ACCCAGGG570
322 GCCCUGGGCUGAUGAGX CGAA AAGAAUGU43 ACATTCTTA CCCAGGGC571
334 GWAGGCA CUGAUGAGX CGAA AGGGCCCU44 AGGGCCCTT TGCCTAAC572
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Table 3
335 UGUUAGGC CUGAUGAG X CGAA AAGGGCCC45 GGGCCCTT T GCCTAACA573
340. GCAUGUGU CUGAUGAG X CGAA AGGCAAAG46 CTTTGCCT A ACACATGC574
352 CCAAAAGU CUGAUGAG X CGAA ACCGCAUG47 CATGCGGT C ACTTTTGG575
356 UCUCCCAA CUGAUGAG X CGAA AGUGACCG48 CGGTCACT T TTGGGAGA576
357 AUCUCCCA CUGAUGAG X CGAA AAGUGACC49 GGTCACTT T TGGGAGAT577
358 CAUCUCCC CUGAUGAG X CGAA AAAGUGAC50 GTCACTTT T GGGAGATG578
393 AGCAUGAC CUGAUGAG X CGAA ACACCCCU51 AGGGGTGT C GTCATGCT579
396 UUGAGCAU CUGAUGAG X CGAA ACGACACC52 GGTGTCGT C ATGCTCAA580
402 ACUCUGUU CUGAUGAG X CGAA AGCAUGAC53 GTCATGCT C AACAGAGT581
424 UUUUAACG CUGAUGAG X CGAA ACCUUUCU54 AGAAAGGT T CGTTAAAA582
425 AUUUUAAC CUGAUGAG X CGAA AACCUUUC55 GAAAGGTT C GTTAAAAT583
428 CGCAUUUU CUGAUGAG X CGAA ACGAACCU56 AGGTTCGT T AAAATGCG584
429 GCGCAUUU CUGAUGAG X CGAA AACGAACC57 GGTTCGTT A AAATGCGC585
443 GUGGCCAG CUGAUGAG X CGAA AUUGUGCG58 CGCACAAT A CTGGCCAC586
474 UCUUCAAA CUGAUGAG X CGAA AUCAUCUC59 GAGATGAT C TTTGAAGA587
476 UGUCUUCA CUGAUGAG X CGAA AGAUCAUC60 GATGATCT T TGAAGACA588
477 GUGUCUUC CUGAUGAG X CGAA AAGAUCAU61 ATGATCTT T GAAGACAC589
490 UAAUUUCA CUGAUGAG X CGAA AUUUGUGU62 ACACAAAT T TGAAATTA590
491 UUAAUUUC CUGAUGAG X CGAA AAUUUGUG63 CACAAATT T GAAATTAA591
497 UCAAUGUU CUGAUGAG X CGAA AUUUCAAA64 TTTGAAAT T AACATTGA592
498 AUCAAUGU CUGAUGAG X CGAA AAUUUCAA65 TTGAAATT A ACATTGAT593
503 CAGAGAUC CUGAUGAG X CGAA AUGUUAAU66 ATTAACAT T GATCTCTG594
507 UCUUCAGA CUGAUGAG X CGAA AUCAAUGU67 ACATTGAT C TCTGAAGA595
509 UAUCUUCA CUGAUGAG X CGAA AGAUCAAU68 ATTGATCT C TGAAGATA596
517 UGACUUGA CUGAUGAG X CGAA AUCUUCAG69 CTGAAGAT A TCAAGTCA597
519 UAUGACUU CUGAUGAG X CGAA AUAUCUUC70 GAAGATAT C AAGTCATA598
524 UAUAAUAU CUGAUGAG X CGAA ACUUGAUA71 TATCAAGT C ATATTATA599
527 CUGUAUAA CUGAUGAG X CGAA AUGACUUG72 CAAGTCAT A TTATACAG600
529 CACUGUAU CUGAUGAG X CGAA AUAUGACU73 AGTCATAT T ATACAGTG601
530 GCACUGUA CUGAUGAG X CGAA AAUAUGAC74 GTCATATT A TACAGTGC602
532 UCGCACUG CUGAUGAG X CGAA AUAAUAUG75 CATATTAT A CAGTGCGA603
546 UCCAAUUC CUGAUGAG X CGAA AGCUGUCG76 CGACAGCT A GAATTGGA604
551 GGUUUUCC CUGAUGAG X CGAA AUUCUAGC77 GCTAGAAT T GGAAAACC605
561 UGGGUUGU CUGAUGAG X CGAA AGGUUUUC78 GAAAACCT T ACAACCCA606
562 UUGGGUUG CUGAUGAG X CGAA AAGGUUUU79 AAAACCTT A CAACCCAA607
577 GAUCUCUC CUGAUGAG X CGAA AGUUUCUU80 AAGAAACT C GAGAGATC608
585 AAAUGUAA CUGAUGAG X CGAA AUCUCUCG81 CGAGAGAT C TTACATTT609
587 GGAAAUGU CUGAUGAG X CGAA AGAUCUCU82 AGAGATCT T ACATTTCC610
588 UGGAAAUG CUGAUGAG X CGAA AAGAUCUC83 GAGATCTT A CATTTCCA611
592 AUAGUGGA CUGAUGAG X CGAA AUGUAAGA84 TCTTACAT T TCCACTAT612
593 UAUAGUGG CUGAUGAG X CGAA AAUGUAAG85 CTTACATT T CCACTATA613
594 GUAUAGUG CUGAUGAG X CGAA AAAUGUAA86 TTACATTT C CACTATAC614
599 AUGUGGUA CUGAUGAG X CGAA AGUGGAAA87 TTTCCACT A TACCACAT615
601 CCAUGUGG CUGAUGAG X CGAA AUAGUGGA88 TCCACTAT A CCACATGG616
617 GGACUCCA CUGAUGAG X CGAA AGUCAGGC89 GCCTGACT T TGGAGTCC617
618 GGGACUCC CUGAUGAG X CGAA AAGUCAGG90 CCTGACTT T GGAGTCCC618
624 GAUUCAGG CUGAUGAG X CGAA ACUCCAAA91 TTTGGAGT C CCTGAATC619
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632 AGGCUGGU CUGAUGAG X CGAA AUUCAGGG92 CCCTGAAT C ACCAGCCT620
641 UCAAGAAU CUGAUGAG X CGAA AGGCUGGU93 ACCAGCCT C ATTCTTGA621
644 AGUUCAAG CUGAUGAG X CGAA AUGAGGCU94 AGCCTCAT T CTTGAACT622
645 AAGUUCAA CUGAUGAG X CGAA AAUGAGGC95 GCCTCATT C TTGAACTT623
647 GAAAGUUC CUGAUGAG X CGAA AGAAUGAG96 CTCATTCT T GAACTTTC624
653 UGAAAAGA CUGAUGAG X CGAA AGUUCAAG97 CTTGAACT T TCTTTTCA625
654 UUGAAAAG CUGAUGAG X CGAA AAGUUCAA98 TTGAACTT T CTTTTCAA626
655 UUUGAAAA CUGAUGAG X CGAA AAAGUUCA99 TGAACTTT C TTTTCAAA627
657 ACUUUGAA CUGAUGAG X CGAA AGAAAGUU100 AACTTTCT T TTCAAAGT628
658 GACUUUGA CUGAUGAG X CGAA AAGAAAGU101 ACTTTCTT T TCAAAGTC629
659 GGACUUUG CUGAUGAG X CGAA AAAGAAAG102 CTTTCTTT T CAAAGTCC630
660 CGGACUUU CUGAUGAG X CGAA AAAAGAAA103 TTTCTTTT C AAAGTCCG631
666 GACUCUCG CUGAUGAG X CGAA ACUUUGAA104 TTCAAAGT C CGAGAGTC632
674 GUGACCCU CUGAUGAG X CGAA ACUCUCGG105 CCGAGAGT C AGGGTCAC633
680 GGCUGAGU CUGAUGAG X CGAA ACCCUGAC106 GTCAGGGT C ACTCAGCC634
684 UCCGGGCU CUGAUGAG X CGAA AGUGACCC107. GGGTCACT C AGCCCGGA635
705 UGCACCAC CUGAUGAG X CGAA ACGGGCCC108 GGGCCCGT T GTGGTGCA636
729 GACCUGCC CUGAUGAG X CGAA AUGCCUGC109 GCAGGCAT C GGCAGGTC637
737 AGGUUCCA CUGAUGAG X CGAA ACCUGCCG110 CGGCAGGT C TGGAACCT638
746 CCAGACAG CUGAUGAG X CGAA AGGUUCCA111 TGGAACCT T CTGTCTGG639
747 GCCAGACA CUGAUGAG X CGAA AAGGUUCC112 GGAACCTT C TGTCTGGC640
751 AUCAGCCA CUGAUGAG X CGAA ACAGAAGG113 CCTTCTGT C TGGCTGAT641
760 GAGGCAGG CUGAUGAG X CGAA AUCAGCCA114 TGGCTGAT A CCTGCCTC642
768 AUCAGCAA CUGAUGAG X CGAA AGGCAGGU115 ACCTGCCT C TTGCTGAT643
770 CCAUCAGC CUGAUGAG X CGAA AGAGGCAG116 CTGCCTCT T GCTGATGG644
796 AACGGAAG CUGAUGAG X CGAA AGGGUCUU117 AAGACCCT T CTTCCGTT645
797 CAACGGAA CUGAUGAG X CGAA AAGGGUCU118 AGACCCTT C TTCCGTTG646
799 AUCAACGG CUGAUGAG X CGAA AGAAGGGU119 ACCCTTCT T CCGTTGAT647
800 UAUCAACG CUGAUGAG X CGAA AAGAAGGG120 CCCTTCTT C CGTTGATA648
804 UUGAUAUC CUGAUGAG X CGAA ACGGAAGA121 TCTTCCGT T GATATCAA649
808 UUUCUUGA CUGAUGAG X CGAA AUCAACGG122 CCGTTGAT A TCAAGAAA650
-
810 ACUUUCUU CUGAUGAG X CGAA AUAUCAAC123 GTTGATAT C AAGAAAGT651
824 UCAUUUCU CUGAUGAG X CGAA ACAGCACU124 AGTGCTGT T AGAAATGA652
825 CUCAUUUC CUGAUGAG X CGAA AACAGCAC125 GTGCTGTT A GAAATGAG653
839 CCAUCCGA CUGAUGAG X CGAA ACUUCCUC126 GAGGAAGT T TCGGATGG654
840 CCCAUCCG CUGAUGAG X CGAA AACUUCCU127 AGGAAGTT T CGGATGGG655
841 CCCCAUCC CUGAUGAG X CGAA AAACUUCC128 GGAAGTTT C GGATGGGG656
855 GCUGUCUG CUGAUGAG X CGAA AUCAGCCC129 GGGCTGAT C CAGACAGC657
878 GGUAGGAG CUGAUGAG X CGAA AGCGCAGC130 GCTGCGCT T CTCCTACC658
,
879 AGGUAGGA CUGAUGAG X CGAA AAGCGCAG131 CTGCGCTT C TCCTACCT659
881 CCAGGUAG CUGAUGAG X CGAA AGAAGCGC132 GCGCTTCT C CTACCTGG660
884 CAGCCAGG CUGAUGAG X CGAA AGGAGAAG133 CTTCTCCT A CCTGGCTG661
897 GCACCUUC CUGAUGAG X CGAA AUCACAGC134 GCTGTGAT C GAAGGTGC662
911 CCAUGAUG CUGAUGAG X CGAA AUUUGGCA135 TGCCAAAT T CATCATGG663
912 CCCAUGAU CUGAUGAG X CGAA AAUUUGGC136 GCCAAATT C ATCATGGG664
915 UCCCCCAU CUGAUGAG X CGAA AUGAAUUU137 AAATTCAT C ATGGGGGA665
926 GCACGGAA CUGAUGAG X CGAA AGUCCCCC138 GGGGGACT C TTCCGTGC666
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928 CUGCACGGCUGAUGAGX CGAA AGAGUCCC139 GGGACTCT T CCGTGCAG667
929 CCUGCACGCUGAUGAGX CGAA AAGAGUCC140 GGACTCTT C CGTGCAGG668
940 CUUCCACUCUGAUGAGX CGAA AUCCUGCA141 TGCAGGAT C AGTGGAAG669
954 UCGUGGGACUGAUGAGX CGAA AGCUCCUU142 AAGGAGCT T TCCCACGA670
955 CUCGUGGGCUGAUGAGX CGAA AAGCUCCU143 AGGAGCTT T CCCACGAG671
956 CCUCGUGGCUGAUGAGX CGAA AAAGCUCC144 GGAGCTTT C CCACGAGG672
988 UGGGGGGACUGAUGAGX CGAA AUGCUCGG145 CCGAGCAT A TCCCCCCA673
990 GGUGGGGGCUGAUGAGX CGAA AUAUGCUC146 GAGCATAT C CCCCCACC674
1000 UGGCCGGGCUGAUGAGX CGAA AGGUGGGG147 CCCCACCT C CCCGGCCA675
1020 GGCUCCAGCUGAUGAGX CGAA AUUCGUUU148 AAACGAAT C CTGGAGCC676
1052 UUGGGAAGCUGAUGAGX CGAA ACUCCCUG149 CAGGGAGT T CTTCCCAA677
1053 UUUGGGAACUGAUGAGX CGAA AACUCCCU150 AGGGAGTT C TTCCCAAA678
1055 GAUUUGGGCUGAUGAGX CGAA AGAACUCC151 GGAGTTCT T CCCAAATC679
1056 UGAUUUGGCUGAUGAGX CGAA AAGAACUC152 GAGTTCTT C CCAAATCA680
1063 CCACUGGUCUGAUGAGX CGAA AUUUGGGA153 TCCCAAAT C ACCAGTGG681
1096 GCAGUCUUCUGAUGAGX CGAA AUCCUCCU154 AGGAGGAT A 682
AAGACTGC
1110 UCUUCCUUCUGAUGAGX CGAA AUGGGGCA155 TGCCCCAT C 683
AAGGAAGA
1133 CGGCAUUU X CGAA AGGGGCUU156 AAGCCCCT T 684
CUGAUGAG AAATGCCG
1134 GCGGCAUUCUGAUGAGX CGAA AAGGGGCU157 AGCCCCTT A 685
AATGCCGC
1148 CGAUGCCGCUGAUGAGX CGAA AGGGUGCG158 CGCACCCT A CGGCATCG686
1155 AUGCUUUCCUGAUGAGX CGAA AUGCCGUA159 TACGGCAT C GAAAGCAT687
1168 AGUGUCUU X CGAA ACUCAUGC160 GCATGAGT C 688
CUGAUGAG AAGACACT
1182 CGACUUCU X CGAA ACUUCAGU161 ACTGAAGT T AGAAGTCG689
CUGAUGAG
1183 CCGACUUCCUGAUGAGX CGAA AACUUCAG162 CTGAAGTT A GAAGTCGG690
1189 CACGACCCCUGAUGAGX CGAA ACUUCUAA163 TTAGAAGT C GGGTCGTG691
1194 CCCCCCACCUGAUGAGX CGAA ACCCGACU164 AGTCGGGT C GTGGGGGG692
1207 ACCUCGAA X CGAA ACUUCCCC165 GGGGAAGT C TTCGAGGT693
CUGAUGAG
1209 GCACCUCGCUGAUGAGX CGAA AGACUUCC166 GGAAGTCT T CGAGGTGC694
1210 GGCACCUCCUGAUGAGX CGAA AAGACUUC167 GAAGTCTT C GAGGTGCC695
1229 UGGCUGGGCUGAUGAGX CGAA AGGCAGCC168 GGCTGCCT C CCCAGCCA696
1250 CGGGCAGUCUGAUGAGX CGAA ACGGCUCC169 GGAGCCGT C ACTGCCCG697
1285 CUUCCAGUCUGAUGAGX CGAA ACUCAGUG170 CACTGAGT T ACTGGAAG698
1286 GCUUCCAGCUGAUGAGX CGAA AACUCAGU171 ACTGAGTT A CTGGAAGC699
1298 UGACCAGGCUGAUGAGX CGAA AGGGCUUC172 GAAGCCCT T CCTGGTCA700
1299 UUGACCAGCUGAUGAGX CGAA AAGGGCUU173 AAGCCCTT C CTGGTCAA701
1305 CACAUGUU X CGAA ACCAGGAA174 TTCCTGGT C 702
CUGAUGAG AACATGTG
1321 GAGGACCGCUGAUGAGX CGAA AGCCACGC175 GCGTGGCT A CGGTCCTC703
1326 GCCGUGAGCUGAUGAGX CGAA ACCGUAGC176 GCTACGGT C CTCACGGC704
1329 CCGGCCGUCUGAUGAGX CGAA AGGACCGU177 ACGGTCCT C ACGGCCGG705
1342 GCAGAGGUCUGAUGAGX CGAA AGCGCCGG178 CCGGCGCT T ACCTCTGC706
1343 AGCAGAGGCUGAUGAGX CGAA AAGCGCCG179 CGGCGCTT A CCTCTGCT707
1347 CUGUAGCACUGAUGAGX CGAA AGGUAAGC180 GCTTACCT C TGCTACAG708
1352 GGAACCUGCUGAUGAGX CGAA AGCAGAGG181 CCTCTGCT A CAGGTTCC709
1358 UGAACAGGCUGAUGAGX CGAA ACCUGUAG182 CTACAGGT T CCTGTTCA710
1359 UUGAACAGCUGAUGAGX CGAA AACCUGUA183 TACAGGTT C CTGTTCAA711
1364 UGCUGUUGCUGAUGAGX CGAA ACAGGAAC184 GTTCCTGT T CAACAGCA712
1365 UUGCUGUU X CGAA AACAGGAA185 TTCCTGTT C 713
CUGAUGAG AACAGCAA
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1379 GGUCAGGCCUGAUGAGX CGAA AUGUGUUG186 CAACACAT A GCCTGACC714
1390 GAGUGGAGCUGAUGAGX CGAA AGGGUCAG187 CTGACCCT C CTCCACTC715
1393 GUGGAGUGCUGAUGAGX CGAA AGGAGGGU188 ACCCTCCT C CACTCCAC716
1398 UGGAGGUGCUGAUGAGX CGAA AGUGGAGG189 CCTCCACT C CACCTCCA717
1404 AGUGGGUGCUGAUGAGX CGAA AGGUGGAG190 CTCCACCT C CACCCACT718
1415 CAGAGGCGCUGAUGAGX CGAA ACAGUGGG191 CCCACTGT C CGCCTCTG719
1421 UGCGGGCACUGAUGAGX CGAA AGGCGGAC192 GTCCGCCT C TGCCCGCA720
1446 AUGCCUGCCUGAUGAGX CGAA AGUCGGGC193 GCCCGACT A GCAGGCAT721
1463 CCCUUACCCUGAUGAGX CGAA ACCGCGGC194 GCCGCGGT A GGTAAGGG722
1467 GCGGCCCUCUGAUGAGX CGAA ACCUACCG195 CGGTAGGT A AGGGCCGC723
1486 CGGCUCUCCUGAUGAGX CGAA ACGCGGUC196 GACCGCGT A GAGAGCCG724
1511 GCAGAACCCUGAUGAGX CGAA ACGUCCGU197 ACGGACGT T GGTTCTGC725
1515 UAGUGCAGCUGAUGAGX CGAA ACCAACGU198 ACGTTGGT T CTGCACTA726
1516 UUAGUGCACUGAUGAGX CGAA AACCAACG199 CGTTGGTT C TGCACTAA727
1523 AUGGGUUU X CGAA AGUGCAGA200 TCTGCACT A AAACCCAT728
CUGAUGAG
1532 CCGGGGAACUGAUGAGX CGAA AUGGGUUU201 AAACCCAT C TTCCCCGG729
1534 AUCCGGGGCUGAUGAGX CGAA AGAUGGGU202 ACCCATCT T CCCCGGAT730
1535 CAUCCGGGCUGAUGAGX CGAA AAGAUGGG203 CCCATCTT C CCCGGATG731
1549 AGGGGUGACUGAUGAGX CGAA ACACACAU204 ATGTGTGT C TCACCCCT732
1551 UGAGGGGUCUGAUGAGX CGAA AGACACAC205 GTGTGTCT C ACCCCTCA733
.
1558 AAAAGGAUCUGAUGAGX CGAA AGGGGUGA206 TCACCCCT C ATCCTTTT734
1561 AGUAAAAGCUGAUGAGX CGAA AUGAGGGG207 CCCCTCAT C CTTTTACT735
1564 AAAAGUAA X CGAA AGGAUGAG208 CTCATCCT T TTACTTTT736
CUGAUGAG
1565 AAAAAGUACUGAUGAGX CGAA AAGGAUGA209 TCATCCTT T TACTTTTT737
1566 CAAAAAGUCUGAUGAGX CGAA AAAGGAUG210 CATCCTTT T ACTTTTTG738
1567 GCAAAAAGCUGAUGAGX CGAA AAAAGGAU211 ATCCTTTT A CTTTTTGC739
1570 GGGGCAAA X CGAA AGUAAAAG212 CTTTTACT T TTTGCCCC740
CUGAUGAG
1571 AGGGGCAA X CGAA AAGUAAAA213 TTTTACTT T TTGCCCCT741
CUGAUGAG
1572 AAGGGGCACUGAUGAGX CGAA AAAGUAAA214 TTTACTTT T TGCCCCTT742
1573 GAAGGGGCCUGAUGAGX CGAA AAAAGUAA215 TTACTTTT T GCCCCTTC743
1580 CAAAGUGGCUGAUGAGX CGAA AGGGGCAA216 TTGCCCCT T CCACTTTG744
1581 UCAAAGUGCUGAUGAGX CGAA AAGGGGCA217 TGCCCCTT C CACTTTGA745
1586 GGUACUCACUGAUGAGX CGAA AGUGGAAG218 CTTCCACT T TGAGTACC746
1587 UGGUACUCCUGAUGAGX CGAA AAGUGGAA219 TTCCACTT T GAGTACCA747
1592 GGAUUUGGCUGAUGAGX CGAA ACUCAAAG220 CTTTGAGT A CCAAATCC748
1599 GGCUUGUGCUGAUGAGX CGAA AUUUGGUA221 TACCAAAT C CACAAGCC749
1610 CCUCAAAACUGAUGAGX CGAA AUGGCUUG222 CAAGCCAT T TTTTGAGG750
1611 UCCUCAAA X CGAA AAUGGCUU223 AAGCCATT T TTTGAGGA751
CUGAUGAG
1612 CUCCUCAA X CGAA AAAUGGCU224 AGCCATTT T TTGAGGAG752
CUGAUGAG
1613 UCUCCUCACUGAUGAGX CGAA AAAAUGGC225 GCCATTTT T TGAGGAGA753
1614 CUCUCCUCCUGAUGAGX CGAA AAAAAUGG226 CCATTTTT T GAGGAGAG754
1634 CAGCAUGGCUGAUGAGX CGAA ACUCUCUU227 AAGAGAGT A CCATGCTG755
1665 GACGGGUGCUGAUGAGX CGAA AGGCCCCU228 AGGGGCCT A CACCCGTC756
1673 AGCCCCAA X CGAA ACGGGUGU229 ACACCCGT C TTGGGGCT757
CUGAUGAG
1675 CGAGCCCCCUGAUGAGX CGAA AGACGGGU230 ACCCGTCT T GGGGCTCG758
1682 GGUGGGGCCUGAUGAGX CGAA AGCCCCAA231 TTGGGGCT C GCCCCACC759
1698 CCAGGAGGCUGAUGAGX CGAA AGCCCUGG232 ~ CCAGGGCTC CCTCCTGG760
I I
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1702 UGCUCCAG CUGAUGAG X CGAA AGGGAGCC233 GGCTCCCT C CTGGAGCA761
1712 CCGCCUGG CUGAUGAG X CGAA AUGCUCCA234 TGGAGCAT C CCAGGCGG762
1746 GCAGAWC CUGAUGAG X CGAA AGGGGGGG235 CCCCCCCT T GAATCTGC763
1751 UCCCUGCA CUGAUGAG X CGAA AUUCAAGG236 CCTTGAAT C TGCAGGGA764
1766 GGAGUGGA CUGAUGAG X CGAA AGUUGCUC237 GAGCAACT C TCCACTCC765
1768 AUGGAGUG CUGAUGAG X CGAA AGAGUUGC238 GCAACTCT C CACTCCAT766
1773 UAAAUAUG CUGAUGAG X CGAA AGUGGAGA239 TCTCCACT C CATATTTA767
1777 UAAAUAAA CUGAUGAG X CGAA AUGGAGUG240 CACTCCAT A TTTATTTA768
1779 UUUAAAUA CUGAUGAG X CGAA AUAUGGAG241 CTCCATAT T TATTTAAA769
1780 GUUUAAAU CUGAUGAG X CGAA AAUAUGGA242 TCCATATT T ATTTAAAC770
1781 UGUUUAAA CUGAUGAG X CGAA AAAUAUGG243 CCATATTT A TTTAAACA771
1783 AUUGUWA CUGAUGAG X CGAA AUAAAUAU244 ATATTTAT T TAAACAAT772
1784 AAUUGUUU CUGAUGAG X CGAA AAUAAAUA245 TATTTATT T AAACAATT773
1785 AAAUUGUU CUGAUGAG X CGAA AAAUAAAU246 ATTTATTT A AACAATTT774
1792 GGGGAAAA CUGAUGAG X CGAA AUUGUUUA247 TAAACAAT T TTTTCCCC775
1793 UGGGGAAA CUGAUGAG X CGAA AAUUGUUU248 AAACAATT T TTTCCCCA776
1794 UUGGGGAA CUGAUGAG X CGAA AAAUUGUU249 AACAATTT T TTCCCCAA777
1795 UWGGGGA CUGAUGAG X CGAA AAAAUUGU250 ACAATTTT T TCCCCAAA778
1796 CUUUGGGG CUGAUGAG X CGAA AAAAAUUG251 CAATTTTT T CCCCAAAG779
1797 CCUUUGGG CUGAUGAG X CGAA AAAAAAUU252 AATTTTTT C CCCAAAGG780
1809 GCACUAUG CUGAUGAG X CGAA AUGCCUUU253 AAAGGCAT C CATAGTGC781
1813 UAGUGCAC CUGAUGAG X CGAA AUGGAUGC254 GCATCCAT A GTGCACTA782
1821 GAAAAUGC CUGAUGAG X CGAA AGUGCACU255 AGTGCACT A GCATTTTC783
1826 UUCAAGAA CUGAUGAG X CGAA AUGCUAGU256 ACTAGCAT T TTCTTGAA784
1827 GUUCAAGA CUGAUGAG X CGAA AAUGCUAG257 CTAGCATT T TCTTGAAC785
1828 GGUUCAAG CUGAUGAG X CGAA AAAUGCUA258 TAGCATTT T CTTGAACC786
1829 UGGUUCAA CUGAUGAG X CGAA AAAAUGCU259 AGCATTTT C TTGAACCA787
1831 AUUGGUUC CUGAUGAG X CGAA AGAAAAUG260 CATTTTCT T GAACCAAT788
1840 UAAUACAU CUGAUGAG X CGAA AUUGGUUC261 GAACCAAT A ATGTATTA789
1845 AAUUUUAA CUGAUGAG X CGAA ACAUUAUU262 AATAATGT A TTAAAATT790
1847 AAAAUUUU CUGAUGAG X CGAA AUACAUUA263 TAATGTAT T AAAATTTT791
1848 AAAAAUUU CUGAUGAG X CGAA AAUACAUU264 AATGTATT A AAATTTTT792
1853 CAUCAAAA CUGAUGAG X CGAA AUUUUAAU265 ATTAAAAT T TTTTGATG793
1854 ACAUCAAA CUGAUGAG X CGAA AAUUUUAA266 TTAAAATT T TTTGATGT794
1855 GACAUCAA CUGAUGAG X CGAA AAAUUUUA267 TAAAATTT T TTGATGTC795
1856 UGACAUCA CUGAUGAG X CGAA AAAAUUUU268 AAAATTTT T TGATGTCA796
1857 CUGACAUC CUGAUGAG X CGAA AAAAAUUU269 AAATTTTT T GATGTCAG797
1863 GCAAGGCU CUGAUGAG X CGAA ACAUCAAA270 TTTGATGT C AGCCTTGC798
1869 CUUGAUGC CUGAUGAG X CGAA AGGCUGAC271 GTCAGCCT T GCATCAAG799
1874 AAGCCCUU CUGAUGAG X CGAA AUGCAAGG272 CCTTGCAT C AAGGGCTT800
1882 UUUUGAUA CUGAUGAG X CGAA AGCCCUUG273 CAAGGGCT T TATCAAAA801
1883 UUUUUGAU CUGAUGAG X CGAA AAGCCCUU274 AAGGGCTT T ATCAAAAA802
1884 CUUUUUGA CUGAUGAG X CGAA AAAGCCCU275 AGGGCTTT A TCAAAAAG803
1886 UACUUUW CUGAUGAG X CGAA AUAAAGCC276 GGCTTTAT C AAAAAGTA804
1894 UAUUAUUG CUGAUGAG X CGAA ACUUUUUG277 CAAAAAGT A CAATAATA805
1899 GGAUUUAU CUGAUGAG X CGAA AUUGUACU278 AGTACAAT A ATAAATCC806
1902 UGAGGAUU CUGAUGAG X CGAA AUUAUUGU279 ACAATAAT A AATCCTCA807
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1906 UACCUGAGCUGAUGAGX CGAA AUUUAUUA280 TAATAAAT C CTCAGGTA808
1909 UACUACCUCUGAUGAGX CGAA AGGAUUUA281 TAAATCCT C AGGTAGTA809
1914 CCCAGUACCUGAUGAGX CGAA ACCUGAGG282 CCTCAGGT A GTACTGGG810
1917 AUUCCCAGCUGAUGAGX CGAA ACUACCUG283 CAGGTAGT A CTGGGAAT811
1934 CCAUGGCACUGAUGAGX CGAA AGCCUUCC284 GGAAGGCT T TGCCATGG812
1935 CCCAUGGCCUGAUGAGX CGAA AAGCCWC285 GAAGGCTT T GCCATGGG813
1954 ACUGGUCUCUGAUGAGX CGAA ACGCAGCA286 TGCTGCGT C AGACCAGT814
1963 CUUCCCAGCUGAUGAGX CGAA ACUGGUCU287 AGACCAGT A CTGGGAAG815
1981 CUGCUUACCUGAUGAGX CGAA ACCGUCCU288 AGGACGGT T GTAAGCAG816
1984 CAACUGCUCUGAUGAGX CGAA ACAACCGU289 ACGGTTGT A AGCAGTTG817
1991 UAAAUAACCUGAUGAGX CGAA ACUGCUUA290 TAAGCAGT T GTTATTTA818
1994 CACUAAAUCUGAUGAGX CGAA ACAACUGC291 GCAGTTGT T ATTTAGTG819
1995 UCACUAAA X CGAA AACAACUG292 CAGTTGTT A TTTAGTGA820
CUGAUGAG
1997 UAUCACUACUGAUGAGX CGAA AUAACAAC293 GTTGTTAT T TAGTGATA821
1998 AUAUCACUCUGAUGAGX CGAA AAUAACAA294 TTGTTATT T AGTGATAT822
1999 AAUAUCACCUGAUGAGX CGAA AAAUAACA295 TGTTATTT A GTGATATT823
2005 ACCCACAA X CGAA AUCACUAA296 TTAGTGAT A TTGTGGGT824
CUGAUGAG
2007 UUACCCACCUGAUGAGX CGAA AUAUCACU297 AGTGATAT T GTGGGTAA825
2014 UCUCACGUCUGAUGAGX CGAA ACCCACAA298 TTGTGGGT A ACGTGAGA826
2027 CAUUGUUCCUGAUGAGX CGAA AUCUUCUC299 GAGAAGAT A GAACAATG827
2038 AUAUAUUACUGAUGAGX CGAA AGCAUUGU300 ACAATGCT A TAATATAT828
2040 UUAUAUAUCUGAUGAGX CGAA AUAGCAUU301 AATGCTAT A ATATATAA829
2043 UCAUUAUACUGAUGAGX CGAA AUUAUAGC302 GCTATAAT A TATAATGA830
2045 GUUCAUUACUGAUGAGX CGAA AUAUUAUA303 TATAATAT A TAATGAAC831
2047 GUGUUCAUCUGAUGAGX CGAA AUAUAUUA304 TAATATAT A ATGAACAC832
2062 UUAUUAAA X CGAA ACCCACGU305 ACGTGGGT A TTTAATAA833
CUGAUGAG
2064 UCUUAUUACUGAUGAGX CGAA AUACCCAC306 GTGGGTAT T TAATAAGA834
2065 UUCUUAUU -XCGAA AAUACCCA307 TGGGTATT T 835
CUGAUGAG AATAAGAA
2066 UUUCUUAUCUGAUGAGX CGAA AAAUACCC308 GGGTATTT A ATAAGAAA836
2069 AUGUUUCUCUGAUGAGX CGAA AUUAAAUA309 TATTTAAT A AGAAACAT837
2088 GACAAAGUCUGAUGAGX CGAA AUCUCACA310 TGTGAGAT T ACTTTGTC838
2089 GGACAAAGCUGAUGAGX CGAA AAUCUCAC311 GTGAGATT A CTTTGTCC839
2092 GCGGGACACUGAUGAGX CGAA AGUAAUCU312 AGATTACT T TGTCCCGC840
2093 AGCGGGACCUGAUGAGX CGAA AAGUAAUC313 GATTACTT T GTCCCGCT841
2096 AUAAGCGGCUGAUGAGX CGAA ACAAAGUA314 TACTTTGT C CCGCTTAT842
2102 AGCAGAAUCUGAUGAGX CGAA AGCGGGAC315 GTCCCGCT T ATTCTGCT843
2103 GAGCAGAA X CGAA AAGCGGGA316 TCCCGCTT A TTCTGCTC844
CUGAUGAG
2105 GGGAGCAGCUGAUGAGX CGAA AUAAGCGG317 CCGCTTAT T CTGCTCCC845
2106 AGGGAGCACUGAUGAGX CGAA AAUAAGCG318 CGCTTATT C TGCTCCCT846
2111 AUAACAGGCUGAUGAGX CGAA AGCAGAAU319 ATTCTGCT C CCTGTTAT847
2117 UAGCAGAUCUGAUGAGX CGAA ACAGGGAG320 CTCCCTGT T ATCTGCTA848
2118 CUAGCAGACUGAUGAGX CGAA AACAGGGA321 TCCCTGTT A TCTGCTAG849
2120 AUCUAGCACUGAUGAGX CGAA AUAACAGG322 CCTGTTAT C TGCTAGAT850
2125 ACUAGAUCCUGAUGAGX CGAA AGCAGAUA323 TATCTGCT A GATCTAGT851
2129 GAGAACUACUGAUGAGX CGAA AUCUAGCA324 TGCTAGAT C TAGTTCTC852
2131 UUGAGAACCUGAUGAGX CGAA AGAUCUAG325 CTAGATCT A GTTCTCAA853
2134 UGAUUGAGCUGAUGAGX CGAA ACUAGAUC326 GATCTAGT T CTCAATCA854
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2135 GUGAUUGACUGAUGAGX CGAA AACUAGAU327 ATCTAGTT C TCAATCAC855
2137 CAGUGAUUCUGAUGAGX CGAA AGAACUAG328 CTAGTTCT C 856
AATCACTG
2141 GGAGCAGUCUGAUGAGX CGAA AUUGAGAA329 TTCTCAAT C ACTGCTCC857
2148 ACACGGGGCUGAUGAGX CGAA AGCAGUGA330 TCACTGCT C CCCCGTGT858
2159 CAUUCUAA X CGAA ACACACGG331 CCGTGTGT A TTAGAATG859
CUGAUGAG
2161 UGCAUUCUCUGAUGAGX CGAA AUACACAC332 GTGTGTAT T AGAATGCA860
2162 AUGCAUUCCUGAUGAGX CGAA AAUACACA333 TGTGTATT A GAATGCAT861
2173 GAAGACCUCUGAUGAGX CGAA ACAUGCAU334 ATGCATGT A AGGTCTTC862
2178 CACAAGAA X CGAA ACCUUACA335 TGTAAGGT C TTCTTGTG863
CUGAUGAG
2180 GACACAAGCUGAUGAGX CGAA AGACCUUA336 TAAGGTCT T CTTGTGTC864
2181 GGACACAA X CGAA AAGACCUU337 AAGGTCTT C TTGTGTCC865
CUGAUGAG
2183 CAGGACACCUGAUGAGX CGAA AGAAGACC338 GGTCTTCT T GTGTCCTG866
2188 UUCAUCAGCUGAUGAGX CGAA ACACAAGA339 TCTTGTGT C CTGATGAA867
2201 CAAGCACACUGAUGAGX CGAA AUUUUUCA340 TGAAAAAT A TGTGCTTG868
2208 CUCAUWC CUGAUGAGX CGAA AGCACAUA341 TATGTGCT T GAAATGAG869
2222 AGAGAUCACUGAUGAGX CGAA AGUUUCUC342 GAGAAACT T TGATCTCT870
2223 CAGAGAUCCUGAUGAGX CGAA AAGUUUCU343 AGAAACTT T GATCTCTG871
2227 UAAGCAGACUGAUGAGX CGAA AUCAAAGU344 ACTTTGAT C TCTGCTTA872
2229 AGUAAGCACUGAUGAGX CGAA AGAUCAAA345 TTTGATCT C TGCTTACT873
2234 ACAUUAGUCUGAUGAGX CGAA AGCAGAGA346 TCTCTGCT T ACTAATGT874
2235 CACAUUAGCUGAUGAGX CGAA AAGCAGAG347 CTCTGCTT A CTAATGTG875
2238 GGGCACAUCUGAUGAGX CGAA AGUAAGCA348 TGCTTACT A ATGTGCCC876
2252 UGGACUUGCUGAUGAGX CGAA ACAUGGGG349 CCCCATGT C CAAGTCCA877
2258 GCAGGUUGCUGAUGAGX CGAA ACUUGGAC350 GTCCAAGT C CAACCTGC878
2283 CAUGUAAUCUGAUGAGX CGAA AUCAGGUC351 GACCTGAT C ATTACATG879
2286 AGCCAUGUCUGAUGAGX CGAA AUGAUCAG352 CTGATCAT T ACATGGCT880
2287 CAGCCAUGCUGAUGAGX CGAA AAUGAUCA353 TGATCATT A CATGGCTG881
2300 GGCUUAGGCUGAUGAGX CGAA ACCACAGC354 GCTGTGGT T CCTAAGCC882
2301 AGGCUUAGCUGAUGAGX CGAA AACCACAG355 CTGTGGTT C CTAAGCCT883
2304. AACAGGCUCUGAUGAGX CGAA AGGAACCA356 TGGTTCCT A AGCCTGTT884
2312 ACUUCAGCCUGAUGAGX CGAA ACAGGCUU357 AAGCCTGT T GCTGAAGT885
2321 GCGACAAUCUGAUGAGX CGAA ACUUCAGC358 GCTGAAGT C ATTGTCGC886
2324 UGAGCGACCUGAUGAGX CGAA AUGACUUC359 GAAGTCAT T GTCGCTCA887
2327 UGCUGAGCCUGAUGAGX CGAA ACAAUGAC360 GTCATTGT C GCTCAGCA888
2331 CUAUUGCUCUGAUGAGX CGAA AGCGACAA361 TTGTCGCT C AGCAATAG889
2338 CUGCACCCCUGAUGAGX CGAA AUUGCUGA362 TCAGCAAT A GGGTGCAG890
2348 UCCUGGAA X CGAA ACUGCACC363 GGTGCAGT T TTCCAGGA891
CUGAUGAG
2349 UUCCUGGACUGAUGAGX CGAA AACUGCAC364 GTGCAGTT T TCCAGGAA892
2350 AUUCCUGGCUGAUGAGX CGAA AAACUGCA365 TGCAGTTT T CCAGGAAT893
2351 UAUUCCUGCUGAUGAGX CGAA AAAACUGC366 GCAGTTTT C CAGGAATA894
2359 CAAAUGCCCUGAUGAGX CGAA AUUCCUGG367 CCAGGAAT A GGCATTTG895
2365 AUUAGGCACUGAUGAGX CGAA AUGCCUAU368 ATAGGCAT T TGCCTAAT896
2366 AAUUAGGCCUGAUGAGX CGAA AAUGCCUA369 TAGGCATT T GCCTAATT897
2371 CCAGGAAUCUGAUGAGX CGAA AGGCAAAU370 ATTTGCCT A ATTCCTGG898
2374 AUGCCAGGCUGAUGAGX CGAA AUUAGGCA371 TGCCTAAT T CCTGGCAT899
2375 CAUGCCAGCUGAUGAGX CGAA AAUUAGGC372 GCCTAATT C CTGGCATG900
2389 AGUCACUACUGAUGAGX CGAA AGUGUCAU373 ATGACACT C TAGTGACT901
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2391 GAAGUCACCUGAUGAGX CGAA AGAGUGUC374 GACACTCT A GTGACTTC902
2398 UCACCAGGCUGAUGAGX CGAA AGUCACUA375 TAGTGACT T CCTGGTGA903
2399 CUCACCAGCUGAUGAGX CGAA AAGUCACU376 AGTGACTT C CTGGTGAG904
2419 UGUACCAGCUGAUGAGX CGAA ACAGGCUG377 CAGCCTGT C CTGGTACA905
2425 CCCUGCUGCUGAUGAGX CGAA ACCAGGAC378 GTCCTGGT A CAGCAGGG906
2435 UACAGCAA X CGAA ACCCUGCU379 AGCAGGGT C TTGCTGTA907
CUGAUGAG
2437 GUUACAGCCUGAUGAGX CGAA AGACCCUG380 CAGGGTCT T GCTGTAAC908
2443 GUCUGAGUCUGAUGAGX CGAA ACAGCAAG381 CTTGCTGT A ACTCAGAC909
2447 GAAUGUCUCUGAUGAGX CGAA AGUUACAG382 CTGTAACT C AGACATTC910
2454 ACCCUUGGCUGAUGAGX CGAA AUGUCUGA383 TCAGACAT T CCAAGGGT911
2455 UACCCUUGCUGAUGAGX CGAA AAUGUCUG384 CAGACATT C CAAGGGTA912
2463 GCUUCCCACUGAUGAGX CGAA ACCCUUGG385 CCAAGGGT A TGGGAAGC913
2475 GGUGUGAACUGAUGAGX CGAA AUGGCUUC386 GAAGCCAT A TTCACACC914
2477 GAGGUGUGCUGAUGAGX CGAA AUAUGGCU387 AGCCATAT T CACACCTC915
2478 UGAGGUGUCUGAUGAGX CGAA AAUAUGGC388 GCCATATT C ACACCTCA916
2485 CAGAGCGUCUGAUGAGX CGAA AGGUGUGA389 TCACACCT C ACGCTCTG917
2491 CAUGUCCACUGAUGAGX CGAA AGCGUGAG390 CTCACGCT C TGGACATG918
2502 CUUCCCUACUGAUGAGX CGAA AUCAUGUC391 GACATGAT T TAGGGAAG919
2503 GCUUCCCUCUGAUGAGX CGAA AAUCAUGU392 ACATGATT T AGGGAAGC920
2504 UGCUUCCCCUGAUGAGX CGAA AAAUCAUG393 CATGATTT A GGGAAGCA921
2536 UGAUCCCACUGAUGAGX CGAA AGGUGGGG394 CCCCACCT T TGGGATCA922
2537 CUGAUCCCCUGAUGAGX CGAA AAGGUGGG395 CCCACCTT T GGGATCAG923
2543 CGGAGGCUCUGAUGAGX CGAA AUCCCAAA396 TTTGGGAT C AGCCTCCG924
2549 GAAUGGCGCUGAUGAGX CGAA AGGCUGAU397 ATCAGCCT C CGCCATTC925
2556 CGACUUGGCUGAUGAGX CGAA AUGGCGGA398 TCCGCCAT T CCAAGTCG926
2557 UCGACUUGCUGAUGAGX CGAA AAUGGCGG399 CCGCCATT C CAAGTCGA927
2563 AGAGUGUCCUGAUGAGX CGAA ACUUGGAA400 TTCCAAGT C GACACTCT928
2570 CUCAAGAA X CGAA AGUGUCGA401 TCGACACT C TTCTTGAG929
CUGAUGAG
2572 UGCUCAAGCUGAUGAGX CGAA AGAGUGUC402 GACACTCT T CTTGAGCA930
2573 CUGCUCAA X CGAA AAGAGUGU403 ACACTCTT C TTGAGCAG931
CUGAUGAG
2575 GUCUGCUCCUGAUGAGX CGAA AGAAGAGU404 ACTCTTCT T GAGCAGAC932
2590 CUCUUCCACUGAUGAGX CGAA AUCACGGU405 ACCGTGAT T TGGAAGAG933
2591 UCUCUUCCCUGAUGAGX CGAA AAUCACGG406 CCGTGATT T GGAAGAGA934
2622 GUUUCAAGCUGAUGAGX CGAA AGUGUGGU407 ACCACACT T CTTGAAAC935
2623 UGUUUCAACUGAUGAGX CGAA AAGUGUGG408 CCACACTT C TTGAAACA936
2625 GCUGUUUCCUGAUGAGX CGAA AGAAGUGU409 ACACTTCT T GAAACAGC937
2646 GCCUAAAGCUGAUGAGX CGAA ACCGUCAC410 GTGACGGT C CTTTAGGC938
2649 GCUGCCUACUGAUGAGX CGAA AGGACCGU411 ACGGTCCT T TAGGCAGC939
2650 GGCUGCCUCUGAUGAGX CGAA AAGGACCG412 CGGTCCTT T AGGCAGCC940
2651 AGGCUGCCCUGAUGAGX CGAA AAAGGACC413 GGTCCTTT A GGCAGCCT941
2668 GGGACAGACUGAUGAGX CGAA ACGGCGGC414 GCCGCCGT C TCTGTCCC942
2670 CCGGGACACUGAUGAGX CGAA AGACGGCG415 CGCCGTCT C TGTCCCGG943
2674 UGAACCGGCUGAUGAGX CGAA ACAGAGAC416 GTCTCTGT C CCGGTTCA944
2680 GCAAGGUGCUGAUGAGX CGAA ACCGGGAC417 GTCCCGGT T CACCTTGC945
2681 GGCAAGGUCUGAUGAGX CGAA AACCGGGA418 TCCCGGTT C ACCTTGCC946
2686 CUCUCGGCCUGAUGAGX CGAA AGGUGAAC419 GTTCACCT T GCCGAGAG947
2703 GUGGGGCACUGAUGAGX CGAA ACGCGCCU420 AGGCGCGT C TGCCCCAC948
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2715 CAGGGUUU X CGAA AGGGUGGG421 CCCACCCT C AAACCCTG949
CUGAUGAG
2741 AGAGUCGUCUGAUGAGX CGAA AGCACCAU422 ATGGTGCT C ACGACTCT950
2748 UGCAGGAACUGAUGAGX CGAA AGUCGUGA423 TCACGACT C TTCCTGCA951
2750 UUUGCAGGCUGAUGAGX CGAA AGAGUCGU424 ACGACTCT T CCTGCAAA952
2751 CUUUGCAGCUGAUGAGX CGAA AAGAGUCG425 CGACTCTT C CTGCAAAG953
2774 UUAAUGUGCUGAUGAGX CGAA AGGUCUUC426 GAAGACCT C CACATTAA954
2780 AGCCACUU X CGAA AUGUGGAG427 CTCCACAT T AAGTGGCT955
CUGAUGAG
2781 AAGCCACUCUGAUGAGX CGAA AAUGUGGA428 TCCACATT A AGTGGCTT956
2789 AUGUUAAA X CGAA AGCCACUU429 AAGTGGCT T TTTAACAT957
CUGAUGAG
2790 CAUGUUAA X CGAA AAGCCACU430 AGTGGCTT T TTAACATG958
CUGAUGAG
2791 UCAUGUUACUGAUGAGX CGAA AAAGCCAC431 GTGGCTTT T TAACATGA959
2792 UUCAUGUU X CGAA AAAAGCCA432 TGGCTTTT T AACATGAA960
CUGAUGAG
2793 UUUCAUGUCUGAUGAGX CGAA AAAAAGCC433 GGCTTTTT A ACATGAAA961
2816 UCGGGAGCCUGAUGAGX CGAA ACAGCUGC434 GCAGCTGT A GCTCCCGA962
2820 UAGCUCGGCUGAUGAGX CGAA AGCUACAG435 CTGTAGCT C CCGAGCTA963
2828 CAAGAGAGCUGAUGAGX CGAA AGCUCGGG436 CCCGAGCT A CTCTCTTG964
2831 UGGCAAGACUGAUGAGX CGAA AGUAGCUC437 GAGCTACT C TCTTGCCA965
2833 GCUGGCAA X CGAA AGAGUAGC438 GCTACTCT C TTGCCAGC966
CUGAUGAG
2835 AUGCUGGCCUGAUGAGX CGAA AGAGAGUA439 TACTCTCT T GCCAGCAT967
2844 AAUGUGAACUGAUGAGX CGAA AUGCUGGC440 GCCAGCAT T TTCACATT968
2845 AAAUGUGACUGAUGAGX CGAA AAUGCUGG441 CCAGCATT T TCACATTT969
2846 AAAAUGUGCUGAUGAGX CGAA AAAUGCUG442 CAGCATTT T CACATTTT970
2847 CAAAAUGUCUGAUGAGX CGAA AAAAUGCU443 AGCATTTT C ACATTTTG971
2852 AAAGGCAA X CGAA AUGUGAAA444 TTTCACAT T TTGCCTTT972
CUGAUGAG
2853 GAAAGGCACUGAUGAGX CGAA AAUGUGAA445 TTCACATT T TGCCTTTC973
2854 AGAAAGGCCUGAUGAGX CGAA AAAUGUGA446 TCACATTT T GCCTTTCT974
2859 CCACGAGACUGAUGAGX CGAA AGGCAAAA447 TTTTGCCT T TCTCGTGG975
2860 ACCACGAGCUGAUGAGX CGAA AAGGCAAA448 TTTGCCTT T CTCGTGGT976
2861 UACCACGACUGAUGAGX CGAA AAAGGCAA449 TTGCCTTT C TCGTGGTA977
2863 UCUACCACCUGAUGAGX CGAA AGAAAGGC450 GCCTTTCT C GTGGTAGA978
2869 CUGGCUUCCUGAUGAGX CGAA ACCACGAG451 CTCGTGGT A GAAGCCAG979
2879 UUUCUCUGCUGAUGAGX CGAA ACUGGCUU452 AAGCCAGT A CAGAGAAA980
2889 CACCACAGCUGAUGAGX CGAA AUUUCUCU453 AGAGAAAT T CTGTGGTG981
2890 CCACCACACUGAUGAGX CGAA AAUUUCUC454 GAGAAATT C TGTGGTGG982
2905 ACACCUCGCUGAUGAGX CGAA AUGUUCCC455 GGGAACAT T CGAGGTGT983
2906 GACACCUCCUGAUGAGX CGAA AAUGUUCC456 GGAACATT C GAGGTGTC984
2914 UGCAGGGUCUGAUGAGX CGAA ACACCUCG457 CGAGGTGT C ACCCTGCA985
2928 CCUCACCACUGAUGAGX CGAA AGCUCUGC458 GCAGAGCT A TGGTGAGG986
2944 CUAAGCCUCUGAUGAGX CGAA AUCCACAC459 GTGTGGAT A AGGCTTAG987
2950 UGGCACCUCUGAUGAGX CGAA AGCCUUAU460 ATAAGGCT T AGGTGCCA988
2951 CUGGCACCCUGAUGAGX CGAA AAGCCUUA461 TAAGGCTT A GGTGCCAG989
2965 AGAAUGCUCUGAUGAGX CGAA ACAGCCUG462 CAGGCTGT A AGCATTCT990
2971 CAGCUCAGCUGAUGAGX CGAA AUGCWAC463 GTAAGCAT T CTGAGCTG991
2972 CCAGCUCACUGAUGAGX CGAA AAUGCUUA464 TAAGCATT C TGAGCTGG992
2983 AAAACAACCUGAUGAGX CGAA AGCCAGCU465 AGCTGGCT T GTTGTTTT993
2986 UUAAAAACCUGAUGAGX CGAA ACAAGCCA466 TGGCTTGT T GTTTTTAA994
2989 GACUUAAA X CGAA ACAACAAG467 CTTGTTGT T TTTAAGTC995
CUGAUGAG
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2990 GGACUUAA X CGAA AACAACAA468 TTGTTGTT T TTAAGTCC996
CUGAUGAG
2991 AGGACUUACUGAUGAGX CGAA AAACAACA469 TGTTGTTT T TAAGTCCT997
2992 CAGGACUUCUGAUGAGX CGAA AAAACAAC470 GTTGTTTT T AAGTCCTG998
2993 ACAGGACUCUGAUGAGX CGAA AAAAACAA471 TTGTTTTT A AGTCCTGT999
2997 AUAUACAGCUGAUGAGX CGAA ACUUAAAA472 TTTTAAGT C CTGTATAT1000
3002 CAUACAUACUGAUGAGX CGAA ACAGGACU473 AGTCCTGT A TATGTATG1001
3004 UACAUACACUGAUGAGX CGAA AUACAGGA474 TCCTGTAT A TGTATGTA1002
3008 CUACUACACUGAUGAGX CGAA ACAUAUAC475 GTATATGT A TGTAGTAG1003
3012 CAAACUACCUGAUGAGX CGAA ACAUACAU476 ATGTATGT A GTAGTTTG1004
3015 ACCCAAACCUGAUGAGX CGAA ACUACAUA477 TATGTAGT A GTTTGGGT1005
3018 CACACCCACUGAUGAGX CGAA ACUACUAC478 GTAGTAGT T TGGGTGTG1006
3019 ACACACCCCUGAUGAGX CGAA AACUACUA479 TAGTAGTT T GGGTGTGT1007
3028 ACUAUAUACUGAUGAGX CGAA ACACACCC480 GGGTGTGT A TATATAGT1008
3030 CUACUAUACUGAUGAGX CGAA AUACACAC481 GTGTGTAT A TATAGTAG1009
3032 UGCUACUACUGAUGAGX CGAA AUAUACAC482 GTGTATAT A TAGTAGCA1010
3034 AAUGCUACCUGAUGAGX CGAA AUAUAUAC483 GTATATAT A GTAGCATT1011
3037 UGAAAUGCCUGAUGAGX CGAA ACUAUAUA484 TATATAGT A GCATTTCA1012
3042 CAUUUUGACUGAUGAGX CGAA AUGCUACU485 AGTAGCAT T TCAAAATG1013
3043 CCAUUUUGCUGAUGAGX CGAA AAUGCUAC486 GTAGCATT T CAAAATGG1014
3044 UCCAUUUU X CGAA AAAUGCUA487 TAGCATTT C AAAATGGA1015
CUGAUGAG
3056 UAAACCAGCUGAUGAGX CGAA ACGUCCAU488 ATGGACGT A CTGGTTTA1016
3062 GGAGGUUACUGAUGAGX CGAA ACCAGUAC489 GTACTGGT T TAACCTCC1017
3063 AGGAGGUU X CGAA AACCAGUA490 TACTGGTT T AACCTCCT1018
CUGAUGAG
3064 UAGGAGGUCUGAUGAGX CGAA AAACCAGU491 ACTGGTTT A ACCTCCTA1019
3069 AAGGAUAGCUGAUGAGX CGAA AGGUUAAA492 TTTAACCT C CTATCCTT1020
3072 UCCAAGGACUGAUGAGX CGAA AGGAGGUU493 AACCTCCT A TCCTTGGA1021
3074 UCUCCAAGCUGAUGAGX CGAA AUAGGAGG494 CCTCCTAT C CTTGGAGA1022
3077 UGCUCUCCCUGAUGAGX CGAA AGGAUAGG495 CCTATCCT T GGAGAGCA1023
3093 AAGGUGGACUGAUGAGX CGAA AGCCAGCU496 AGCTGGCT C TCCACCTT1024
3095 ACAAGGUGCUGAUGAGX CGAA AGAGCCAG497 CTGGCTCT C CACCTTGT1025
3101 UGUGUAACCUGAUGAGX CGAA AGGUGGAG498 CTCCACCT T GTTACACA1026
3104 UAAUGUGUCUGAUGAGX CGAA ACAAGGUG499 CACCTTGT T ACACATTA1027
3105 AUAAUGUGCUGAUGAGX CGAA AACAAGGU500 ACCTTGTT A CACATTAT1028
3111 UCUAACAUCUGAUGAGX CGAA AUGUGUAA501 TTACACAT T ATGTTAGA1029
3112 CUCUAACACUGAUGAGX CGAA AAUGUGUA502 TACACATT A TGTTAGAG1030
3116 ACCUCUCUCUGAUGAGX CGAA ACAUAAUG503 CATTATGT T AGAGAGGT1031
3117 UACCUCUCCUGAUGAGX CGAA AACAUAAU504 ATTATGTT A GAGAGGTA1032
3125 CAGCUCGCCUGAUGAGX CGAA ACCUCUCU505 AGAGAGGT A GCGAGCTG1033
3136 ACAUAGCACUGAUGAGX CGAA AGCAGCUC506 GAGCTGCT C TGCTATGT1034
3141 UAAGGACACUGAUGAGX CGAA AGCAGAGC507 GCTCTGCT A TGTCCTTA1035
3145 GGCUUAAGCUGAUGAGX CGAA ACAUAGCA508 TGCTATGT C CTTAAGCC1036
3148 AUUGGCUU X CGAA AGGACAUA509 TATGTCCT T AAGCCAAT1037
CUGAUGAG
3149 UAUUGGCUCUGAUGAGX CGAA AAGGACAU510 ATGTCCTT A AGCCAATA1038
3157 UGAGUAAA X CGAA AUUGGCUU511 AAGCCAAT A TTTACTCA1039
CUGAUGAG
3159 GAUGAGUACUGAUGAGX CGAA AUAUUGGC512 GCCAATAT T TACTCATC1040
3160 UGAUGAGUCUGAUGAGX CGAA AAUAUUGG513 CCAATATT T ACTCATCA1041
3161 CUGAUGAGCUGAUGAGX CGAA AAAUAUUG514 CAATATTT A CTCATCAG1042
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3164 GACCUGAU CUGAUGAG X CGAA 515 TATTTACT C ATCAGGTC1043
AGUAAAUA
3167 AAUGACCU CUGAUGAG X CGAA 516 TTACTCAT C AGGTCATT1044
AUGAGUAA
3172 AAAAUAAU CUGAUGAG X CGAA 517 CATCAGGT C ATTATTTT1045
ACCUGAUG
3175 UAAAAAAU CUGAUGAG X CGAA 518 CAGGTCAT T ATTTTTTA1046
AUGACCUG
3176 GUAAAAAA CUGAUGAG X CGAA 519 AGGTCATT A TTTTTTAC1047
AAUGACCU
3178 WGUAAAA CUGAUGAG X CGAA AUAAUGAC520 GTCATTAT T TTTTACAA1048
3179 AWGUAAA CUGAUGAG X CGAA AAUAAUGA521 TCATTATT T TTTACAAT1049
3180 CAWGUAA CUGAUGAG X CGAA AAAUAAUG522 CATTATTT T TTACAATG1050
3181 CCAWGUA CUGAUGAG X CGAA AAAAUAAU523 ATTATTTT T TACAATGG1051
3182 GCCAWGU CUGAUGAG X CGAA AAAAAUAA524 TTATTTTT T ACAATGGC1052
3183 GGCCAWG CUGAUGAG X CGAA AAAAAAUA525 TATTTTTT A CAATGGCC1053
3199 AAAUGGW CUGAUGAG X CGAA AWCCAUG526 CATGGAAT A AACCATTT1054
3206 UUUGUAAA CUGAUGAG X CGAA 527 TAAACCAT T TTTACAAA1055
AUGGUUUA
3207 UUWGUAA CUGAUGAG X CGAA AAUGGUW528 AAACCATT T TTACAAAA1056
Input Sequence = PTPN1 (Homo sapiens protein tyrosine phosphatase, non-
receptor type 1 (PTPN1)
3215 bp)
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Table 4
Table 4: Human PTP-1 B NCH Ribozyme and Target Sequence
Nt. Ribozyme Seq. Substrate Seq.
Position Sequence ID Sequence ID
Nos. Nos.
13 CCGCUCUA XCGAAICCGCGUC1057 GACGCGGC C TAGAGCGG 1781
CUGAUGAG
14 GCCGCUCUCUGAUGAGXCGAAIGCCGCGU1058 ACGCGGCC T AGAGCGGC 1782
23 GCGCCGUCCUGAUGAGXCGAAICCGCUCU1059 AGAGCGGC A GACGGCGC 1783
32 CGGCCCACCUGAUGAGXCGAAICGCCGUC1060 GACGGCGC A GTGGGCCG 1784
39 UCCUUCUCCUGAUGAGXCGAAICCCACUG1061 CAGTGGGC C GAGAAGGA 1785
53 GCGGCUGCCUGAUGAGXCGAAICGCCUCC1062 GGAGGCGC A GCAGCCGC 1786
'
56 AGGGCGGCCUGAUGAGXCGAAICUGCGCC1063 GGCGCAGC A GCCGCCCT 1787
59 GCCAGGGCCUGAUGAGXCGAAICUGCUGC1064 GCAGCAGC C GCCCTGGC 1788
62 CGGGCCAGCUGAUGAGXCGAAICGGCUGC1065 GCAGCCGC C CTGGCCCG 1789
63 ACGGGCCACUGAUGAGXCGAAIGCGGCUG1066 CAGCCGCC C TGGCCCGT 1790
64 GACGGGCCCUGAUGAGXCGAAIGGCGGCU1067 AGCCGCCC T GGCCCGTC 1791
68 CCAUGACGCUGAUGAGXCGAAICCAGGGC1068 GCCCTGGC C CGTCATGG 1792
69 UCCAUGACCUGAUGAGXCGAAIGCCAGGG1069 CCCTGGCC C GTCATGGA 1793
73 CAUCUCCACUGAUGAGXCGAAIACGGGCC1070 GGCCCGTC A TGGAGATG 1794
98 UGUCGAUCCUGAUGAGXCGAAICUCGAAC1071 GTTCGAGC A GATCGACA 1795
106 CCCGGACUCUGAUGAGXCGAAIUCGAUCU1072 AGATCGAC A AGTCCGGG 1796
111 CAGCUCCCCUGAUGAGXCGAAIACUUGUC1073 GACAAGTC C GGGAGCTG 1797
118 GGCCGCCCCUGAUGAGXCGAAICUCCCGG1074 CCGGGAGC T GGGCGGCC 1798
126 UGGUAAAUCUGAUGAGXCGAA 1075 TGGGCGGC C ATTTACCA 1799
ICCGCCCA
127 CUGGUAAA XCGAAIGCCGCCC1076 GGGCGGCC A TTTACCAG 1800
CUGAUGAG
133 GAUAUCCUCUGAUGAGXCGAAIUAAAUGG1077 CCATTTAC C AGGATATC 1801
134 GGAUAUCCCUGAUGAGXCGAAIGUAAAUG1078 CATTTACC A GGATATCC 1802
142 UUCAUGUCCUGAUGAGXCGAAIAUAUCCU1079 AGGATATC C GACATGAA 1803
146 UGGCUUCACUGAUGAGXCGAAIUCGGAUA1080 TATCCGAC A TGAAGCCA 1804
153 AAGUCACUCUGAUGAGXCGAAICUUCAUG1081 CATGAAGC C AGTGACTT 1805
154 GAAGUCACCUGAUGAGXCGAAIGCUUCAU1082 ATGAAGCC A GTGACTTC 1806
160 ACAUGGGACUGAUGAGXCGAAIUCACUGG1083 CCAGTGAC T TCCCATGT 1807
163 UCUACAUGCUGAUGAGXCGAAIAAGUCAC1084 GTGACTTC C CATGTAGA 1808
164 CUCUACAUCUGAUGAGXCGAAIGAAGUCA1085 TGACTTCC C ATGTAGAG 1809
165 ACUCUACACUGAUGAGXCGAAIGGAAGUC1086 GACTTCCC A TGTAGAGT 1810
177 GGAAGCUU XCGAAICCACUCU1087 AGAGTGGC C AAGCTTCC 1811
CUGAUGAG
178 AGGAAGCUCUGAUGAGXCGAAIGCCACUC1088 GAGTGGCC A AGCTTCCT 1812
182 UCWAGGA CUGAUGAGXCGAAICUUGGCC1089 GGCCAAGC T TCCTAAGA 1813
185 UGWCUUA CUGAUGAGXCGAAIAAGCUUG1090 CAAGCTTC C TAAGAACA 1814
186 UUGUUCUU XCGAA 1091 AAGCTTCC T AAGAACAA 1815
CUGAUGAG IGAAGCUU
193 UCGGUUUU XCGAAIUUCUUAG1092 CTAAGAAC A 1816
CUGAUGAG AAAACCGA
199 CCUAUUUCCUGAUGAGXCGAAIUUUWGU 1093 ACAAAAAC C GAAATAGG 1817
211 GACGUCUCCUGAUGAGXCGAAIUACCUAU1094 ATAGGTAC A GAGACGTC 1818
220 AAAGGGACCUGAUGAGXCGAAIACGUCUC1095 GAGACGTC A GTCCCTTT 1819
224 GGUCAAAGCUGAUGAGXCGAA 1096 CGTCAGTC C CTTTGACC 1820
IACUGACG
225 UGGUCAAA XCGAAIGACUGAC1097 GTCAGTCC C TTTGACCA 1821
CUGAUGAG
226 AUGGUCAA XCGAAIGGACUGA1098 TCAGTCCC T TTGACCAT 1822
CUGAUGAG
232 CCGACUAUCUGAUGAGXCGAAIUCAAAGG1099 CCTTTGAC C ATAGTCGG 1823
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233 UCCGACUA CUGAUGAG X CGAA IGUCAAAG1100 CTTTGACC A TAGTCGGA 1824
248 CUUGAUGU CUGAUGAG X CGAA IUUUAAUC1101 GATTAAAC T ACATCAAG 1825
251 CUUCUUGA CUGAUGAG X CGAA IUAGUUUA1102 TAAACTAC A TCAAGAAG 1826
254 UAUCUUCU CUGAUGAG X CGAA IAUGUAGU1103 ACTACATC A AGAAGATA 1827
268 GUUGAUAU CUGAUGAG X CGAA IUCAUUAU1104 ATAATGAC T ATATCAAC 1828
274 ACUAGCGU CUGAUGAG X CGAA IAUAUAGU1105 ACTATATC A ACGCTAGT 1829
279 AUCAAACU CUGAUGAG X CGAA ICGUUGAU1106 ATCAACGC T AGTTTGAT 1830
303 CUCCUUUG CUGAUGAG X CGAA ICUUCUUC1107 GAAGAAGC C CAAAGGAG 1831
304 ACUCCUUU CUGAUGAG X CGAA IGCUUCUU1108 AAGAAGCC C AAAGGAGT 1832
305 AACUCCUU CUGAUGAG X CGAA IGGCUUCU1109 AGAAGCCC A AAGGAGTT 1833
316 GGUAAGAA CUGAUGAG X CGAA IUAACUCC1110 GGAGTTAC A TTCTTACC 1834
320 CCUGGGUA CUGAUGAG X CGAA IAAUGUAA1111 TTACATTC T TACCCAGG 1835
324 GGGCCCUG CUGAUGAG X CGAA IUAAGAAU1112 ATTCTTAC C CAGGGCCC 1836
325 AGGGCCCU CUGAUGAG X CGAA IGUAAGAA1113 TTCTTACC C AGGGCCCT 1837
326 AAGGGCCC CUGAUGAG X CGAA IGGUAAGA1114 TCTTACCC A GGGCCCTT 1838
331 AGGCAAAG CUGAUGAG X CGAA ICCCUGGG1115 CCCAGGGC C CTTTGCCT 1839
332 UAGGCAAA CUGAUGAG X CGAA IGCCCUGG1116 CCAGGGCC C TTTGCCTA 1840
333 UUAGGCAA CUGAUGAG X CGAA IGGCCCUG1117 CAGGGCCC T TTGCCTAA 1841
338 AUGUGUUA CUGAUGAG X CGAA ICAAAGGG1118 CCCTTTGC C TAACACAT 1842
339 CAUGUGUU CUGAUGAG X CGAA IGCAAAGG1119 CCTTTGCC T AACACATG 1843
343 ACCGCAUG CUGAUGAG X CGAA IUUAGGCA1120 TGCCTAAC A CATGCGGT 1844
345 UGACCGCA CUGAUGAG X CGAA IUGUUAGG1121 CCTAACAC A TGCGGTCA 1845
353 CCCAAAAG CUGAUGAG X CGAA IACCGCAU1122 ATGCGGTC A CTTTTGGG 1846
355 CUCCCAAA CUGAUGAG X CGAA IUGACCGC1123 GCGGTCAC T TTTGGGAG 1847
377 UGCUUUUC CUGAUGAG X CGAA ICUCCCAC1124 GTGGGAGC A GAAAAGCA 1848
385 GACACCCC CUGAUGAG X CGAA ICUUUUCU1125 AGAAAAGC A GGGGTGTC 1849
397 GUUGAGCA CUGAUGAG X CGAA IACGACAC1126 GTGTCGTC A TGCTCAAC 1850
401 CUCUGUUG CUGAUGAG X CGAA ICAUGACG1127 CGTCATGC T CAACAGAG 1851
403 CACUCUGU CUGAUGAG X CGAA IAGCAUGA1128 TCATGCTC A ACAGAGTG 1852
406 CAUCACUC CUGAUGAG X CGAA IUUGAGCA1129 TGCTCAAC A GAGTGATG 1853
438 CAGUAUUG CUGAUGAG X CGAA ICGCAUUU1130 AAATGCGC A CAATACTG 1854
440 GCCAGUAU CUGAUGAG X CGAA IUGCGCAU1131 ATGCGCAC A ATACTGGC 1855
445 UUGUGGCC CUGAUGAG X CGAA IUAUUGUG1132 CACAATAC T GGCCACAA 1856
449 CUUUUUGU CUGAUGAG X CGAA ICCAGUAU1133 ATACTGGC C ACAAAAAG 1857
450 UCUUUUUG CUGAUGAG X CGAA IGCCAGUA1134 TACTGGCC A CAAAAAGA 1858
452 CUUCUUUU CUGAUGAG X CGAA IUGGCCAG1135 CTGGCCAC A AAAAGAAG 1859
475 GUCUUCAA CUGAUGAG X CGAA IAUCAUCU1136 AGATGATC T TTGAAGAC 1860
484 CAAAUUUG CUGAUGAG X CGAA IUCUUCAA1137 TTGAAGAC A CAAATTTG 1861
486 UUCAAAUU CUGAUGAG X CGAA IUGUCUUC1138 GAAGACAC A AATTTGAA 1862
501 GAGAUCAA CUGAUGAG X CGAA IUUAAUUU1139 AAATTAAC A TTGATCTC 1863
508 AUCUUCAG CUGAUGAG X CGAA IAUCAAUG1140 CATTGATC T CTGAAGAT 1864
510 AUAUCUUC CUGAUGAG X CGAA IAGAUCAA1141 TTGATCTC T GAAGATAT 1865
520 AUAUGACU CUGAUGAG X CGAA IAUAUCUU1142 AAGATATC A AGTCATAT 1866
525 GUAUAAUA CUGAUGAG X CGAA IACUUGAU1143 ATCAAGTC A TATTATAC 1867
534 UGUCGCAC CUGAUGAG X CGAA IUAUAAUA1144 TATTATAC A GTGCGACA 1868
542 AUUCUAGC CUGAUGAG X CGAA IUCGCACU1145 AGTGCGAC A GCTAGAAT 1869
545 CCAAUUCU CUGAUGAG X CGAA ICUGUCGC1146 GCGACAGC T AGAATTGG 1870
1
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Table 4
559 GGUUGUAA X IUUUUCCA1147 TGGAAAAC C TTACAACC 1871
CUGAUGAG CGAA
560 GGGUUGUACUGAUGAGX IGUUUUCC1148 GGAAAACC T TACAACCC 1872
CGAA
564 UCUUGGGUCUGAUGAGX IUAAGGUU1149 AACCTTAC A ACCCAAGA 1873
CGAA
567 GUUUCUUGCUGAUGAGX 1150 CTTACAAC C CAAGAAAC 1874
CGAA
IUUGUAAG
568 AGUUUCUU X IGUUGUAA1151 TTACAACC C 1875
CUGAUGAG CGAA AAGAAACT
569 GAGUUUCUCUGAUGAGX IGGUUGUA1152 TACAACCC A AGAAACTC 1876
CGAA
576 AUCUCUCGCUGAUGAGX IUUUCUUG1153 CAAGAAAC T CGAGAGAT 1877
CGAA
586 GAAAUGUACUGAUGAGX IAUCUCUC1154 GAGAGATC T TACATTTC 1878
CGAA
590 AGUGGAAA X IUAAGAUC1155 GATCTTAC A TTTCCACT 1879
CUGAUGAG CGAA
595 GGUAUAGUCUGAUGAGX IAAAUGUA1156 TACATTTC C ACTATACC 1880
CGAA
596 UGGUAUAGCUGAUGAGX IGAAAUGU1157 ACATTTCC A CTATACCA 1881
CGAA
598 UGUGGUAUCUGAUGAGX IUGGAAAU1158 ATTTCCAC T ATACCACA 1882
CGAA
603 GGCCAUGUCUGAUGAGX IUAUAGUG1159 CACTATAC C ACATGGCC 1883
CGAA
604 AGGCCAUGCUGAUGAGX IGUAUAGU1160 ACTATACC A CATGGCCT 1884
CGAA
606 UCAGGCCACUGAUGAGX IUGGUAUA1161 TATACCAC A TGGCCTGA 1885
CGAA
611 CAAAGUCACUGAUGAGX 1162 CACATGGC C TGACTTTG 1886
CGAA
ICCAUGUG
612 CCAAAGUCCUGAUGAGX 1163 ACATGGCC T GACTTTGG 1887
CGAA
IGCCAUGU
616 GACUCCAA X IUCAGGCC1164 GGCCTGAC T TTGGAGTC 1888
CUGAUGAG CGAA
625 UGAUUCAGCUGAUGAGX IACUCCAA1165 TTGGAGTC C CTGAATCA 1889
CGAA
626 GUGAUUCACUGAUGAGX IGACUCCA1166 TGGAGTCC C TGAATCAC 1890
CGAA
627 GGUGAUUCCUGAUGAGX IGGACUCC1167 GGAGTCCC T GAATCACC 1891
CGAA
633 GAGGCUGGCUGAUGAGX IAUUCAGG1168 CCTGAATC A CCAGCCTC 1892
CGAA
635 AUGAGGCUCUGAUGAGX IUGAUUCA1169 TGAATCAC C AGCCTCAT 1893
CGAA
636 AAUGAGGCCUGAUGAGX 1170 GAATCACC A GCCTCATT 1894
CGAA
IGUGAUUC
639 AAGAAUGACUGAUGAGX ICUGGUGA1171 TCACCAGC C TCATTCTT 1895
CGAA
640 CAAGAAUGCUGAUGAGX IGCUGGUG1172 CACCAGCC T CATTCTTG 1896
CGAA
642 UUCAAGAA X 1173 CCAGCCTC A TTCTTGAA 1897
CUGAUGAG CGAA
IAGGCUGG
646 AAAGUUCACUGAUGAGX 1174 CCTCATTC T TGAACTTT 1898
CGAA
IAAUGAGG
652 GAAAAGAA X IUUCAAGA1175 TCTTGAAC T TTCTTTTC 1899
CUGAUGAG CGAA
656 CUUUGAAA X 1176 GAACTTTC T TTTCAAAG 1900
CUGAUGAG CGAA
IAAAGUUC
661 UCGGACUU X IAAAAGAA1177 TTCTTTTC A AAGTCCGA 1901
CUGAUGAG CGAA
667 UGACUCUCCUGAUGAGX IACUUUGA1178 TCAAAGTC C GAGAGTCA 1902
CGAA
675 AGUGACCCCUGAUGAGX 1179 CGAGAGTC A GGGTCACT 1903
CGAA
IACUCUCG
681 GGGCUGAGCUGAUGAGX 1180 TCAGGGTC A CTCAGCCC 1904
CGAA
IACCCUGA
683 CCGGGCUGCUGAUGAGX 1181 AGGGTCAC T CAGCCCGG 1905
CGAA
IUGACCCU
685 CUCCGGGCCUGAUGAGX IAGUGACC1182 GGTCACTC A GCCCGGAG 1906
CGAA
688 GUGCUCCGCUGAUGAGX ICUGAGUG1183 CACTCAGC C CGGAGCAC 1907
CGAA
689 CGUGCUCCCUGAUGAGX IGCUGAGU1184 ACTCAGCC C GGAGCACG 1908
CGAA
695 CGGGCCCGCUGAUGAGX ICUCCGGG1185 CCCGGAGC A CGGGCCCG 1909
CGAA
701 CCACAACGCUGAUGAGX ICCCGUGC1186 GCACGGGC C CGTTGTGG 1910
CGAA
702 ACCACAACCUGAUGAGX IGCCCGUG1187 CACGGGCC C GTTGTGGT 1911
CGAA
713 CACUGCAGCUGAUGAGX ICACCACA1188 TGTGGTGC A CTGCAGTG 1912
CGAA
715 UGCACUGCCUGAUGAGX IUGCACCA1189 TGGTGCAC T GCAGTGCA 1913
CGAA
718 GCCUGCACCUGAUGAGX ICAGUGCA1190 TGCACTGC A GTGCAGGC 1914
CGAA
723 CCGAUGCCCUGAUGAGX 1191 TGCAGTGC A GGCATCGG 1915
CGAA
ICACUGCA
727 CCUGCCGACUGAUGAGX 1192 GTGCAGGC A TCGGCAGG 1916
CGAA
ICCUGCAC
733 UCCAGACCCUGAUGAGX ICCGAUGC1193 GCATCGGC A GGTCTGGA 1917
CGAA
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738 AAGGUUCCCUGAUGAGXCGAAIACCUGCC1194 GGCAGGTC T GGAACCTT 1918
744 AGACAGAA XCGAAIUUCCAGA1195 TCTGGAAC C TTCTGTCT 1919
CUGAUGAG
745 CAGACAGACUGAUGAGXCGAAIGUUCCAG1196 CTGGAACC T TCTGTCTG 1920
748 AGCCAGACCUGAUGAGXCGAAIAAGGUUC1197 GAACCTTC T GTCTGGCT 1921
752 UAUCAGCCCUGAUGAGXCGAAIACAGAAG1198 CTTCTGTC T GGCTGATA 1922
756 CAGGUAUCCUGAUGAGXCGAAICCAGACA1199 TGTCTGGC T GATACCTG 1923
762 AAGAGGCACUGAUGAGXCGAAIUAUCAGC1200 GCTGATAC C TGCCTCTT 1924
763 CAAGAGGCCUGAUGAGXCGAAIGUAUCAG1201 CTGATACC T GCCTCTTG 1925
766 CAGCAAGACUGAUGAGXCGAAICAGGUAU1202 ATACCTGC C TCTTGCTG 1926
~
767 UCAGCAAGCUGAUGAGXCGAAIGCAGGUA1203 TACCTGCC T CTTGCTGA 1927
769 CAUCAGCACUGAUGAGXCGAAIAGGCAGG1204 CCTGCCTC T TGCTGATG 1928
773 UGUCCAUCCUGAUGAGXCGAAICAAGAGG1205 CCTCTTGC T GATGGACA 1929
781 UUUCCUCUCUGAUGAGXCGAAIUCCAUCA1206 TGATGGAC A AGAGGAAA 1930
793 GGAAGAAGCUGAUGAGXCGAAIUCUUUCC1207 GGAAAGAC C CTTCTTCC 1931
794 CGGAAGAA XCGAAIGUCUUUC1208 GAAAGACC C TTCTTCCG 1932
CUGAUGAG
795 ACGGAAGACUGAUGAGXCGAAIGGUCUUU1209 AAAGACCC T TCTTCCGT 1933
798 UCAACGGACUGAUGAGXCGAAIAAGGGUC1210 GACCCTTC T TCCGTTGA 1934
801 AUAUCAACCUGAUGAGXCGAAIAAGAAGG1211 CCTTCTTC C GTTGATAT 1935
811 CACUUUCUCUGAUGAGXCGAAIAUAUCAA1212 TTGATATC A AGAAAGTG 1936
821 UUUCUAACCUGAUGAGXCGAAICACUUUC1213 GAAAGTGC T GTTAGAAA 1937
851 UCUGGAUCCUGAUGAGXCGAA.ICCCCAUC1214 GATGGGGC T GATCCAGA 1938
856 GGCUGUCUCUGAUGAGXCGAAIAUCAGCC1215 GGCTGATC C AGACAGCC 1939
857 CGGCUGUCCUGAUGAGXCGAAIGAUCAGC1216 GCTGATCC A GACAGCCG 1940
861 UGGUCGGCCUGAUGAGXCGAAIUCUGGAU1217 ATCCAGAC A GCCGACCA 1941
864 AGCUGGUCCUGAUGAGXCGAAICUGUCUG1218 CAGACAGC C GACCAGCT 1942
868 GCGCAGCUCUGAUGAGXCGAAIUCGGCUG1219 CAGCCGAC C AGCTGCGC 1943
869 AGCGCAGCCUGAUGAGXCGAAIGUCGGCU1220 AGCCGACC A GCTGCGCT 1944
872 AGAAGCGCCUGAUGAGXCGAAICUGGUCG1221 CGACCAGC T GCGCTTCT 1945
877 GUAGGAGACUGAUGAGXCGAAICGCAGCU1222 AGCTGCGC T TCTCCTAC 1946
880 CAGGUAGGCUGAUGAGXCGAAIAAGCGCA1223 TGCGCTTC T CCTACCTG 1947
882 GCCAGGUACUGAUGAGXCGAAIAGAAGCG1224 CGCTTCTC C TACCTGGC 1948
883 AGCCAGGUCUGAUGAGXCGAAIGAGAAGC1225 GCTTCTCC T ACCTGGCT 1949
886 CACAGCCACUGAUGAGXCGAAIUAGGAGA1226 TCTCCTAC C TGGCTGTG 1950
887 UCACAGCCCUGAUGAGXCGAAIGUAGGAG1227 CTCCTACC T GGCTGTGA 1951
891 UCGAUCACCUGAUGAGXCGAAICCAGGUA1228 TACCTGGC T GTGATCGA 1952
906 AUGAAUUU XCGAAICACCUUC1229 GAAGGTGC C AAATTCAT 1953
CUGAUGAG
907 GAUGAAUU XCGAAIGCACCUU1230 AAGGTGCC A 1954
CUGAUGAG AATTCATC
913 CCCCAUGACUGAUGAGXCGAAIAAUUUGG1231 CCAAATTC A TCATGGGG 1955
916 GUCCCCCACUGAUGAGXCGAAIAUGAAUU1232 AATTCATC A TGGGGGAC 1956
925 CACGGAAGCUGAUGAGXCGAAIUCCCCCA1233 TGGGGGAC T CTTCCGTG 1957
927 UGCACGGACUGAUGAGXCGAAIAGUCCCC1234 GGGGACTC T TCCGTGCA 1958
930 UCCUGCACCUGAUGAGXCGAA 1235 GACTCTTC C GTGCAGGA 1959
IAAGAGUC
935 ACUGAUCCCUGAUGAGXCGAAICACGGAA1236 TTCCGTGC A GGATCAGT 1960
941 CCUUCCACCUGAUGAGXCGAAIAUCCUGC1237 GCAGGATC A GTGGAAGG 1961
953 CGUGGGAA XCGAA 1238 GAAGGAGC T TTCCCACG 1962
CUGAUGAG ICUCCUUC
957 UCCUCGUGCUGAUGAGXCGAAIAAAGCUC1239 GAGCTTTC C CACGAGGA 1963
958 GUCCUCGUCUGAUGAGXCGAAIGAAAGCU1240 AGCTTTCC C ACGAGGAC 1964
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959 GGUCCUCGCUGAUGAGX IGGAAAGC1241 GCTTTCCC A CGAGGACC 1965
CGAA
967 GGGCUCCACUGAUGAGX IUCCUCGU1242 ACGAGGAC C TGGAGCCC 1966
CGAA
968 GGGGCUCCCUGAUGAGX IGUCCUCG1243 CGAGGACC T GGAGCCCC 1967
CGAA
974 CGGGUGGGCUGAUGAGX ICUCCAGG1244 CCTGGAGC C CCCACCCG 1968
CGAA
975 UCGGGUGGCUGAUGAGX IGCUCCAG1245 CTGGAGCC C CCACCCGA 1969
CGAA
976 CUCGGGUGCUGAUGAGX IGGCUCCA1246 TGGAGCCC C CACCCGAG 1970
CGAA
977 GCUCGGGUCUGAUGAGX IGGGCUCC1247 GGAGCCCC C ACCCGAGC 1971
CGAA
978 UGCUCGGGCUGAUGAGX IGGGGCUC1248 GAGCCCCC A CCCGAGCA 1972
CGAA
980 UAUGCUCGCUGAUGAGX IUGGGGGC1249 GCCCCCAC C CGAGCATA 1973
CGAA
981 AUAUGCUCCUGAUGAGX IGUGGGGG1250 CCCCCACC C GAGCATAT 1974
CGAA
986 GGGGGAUACUGAUGAGX ICUCGGGU1251 ACCCGAGC A TATCCCCC 1975
CGAA
991 AGGUGGGGCUGAUGAGX IAUAUGCU1252 AGCATATC C CCCCACCT 1976
CGAA
992 GAGGUGGGCUGAUGAGX IGAUAUGC1253 GCATATCC C CCCACCTC 1977
CGAA
993 GGAGGUGGCUGAUGAGX 1254 CATATCCC C CCACCTCC 1978
CGAA
IGGAUAUG
994 GGGAGGUGCUGAUGAGX IGGGAUAU1255 ATATCCCC C CACCTCCC 1979
CGAA
995 GGGGAGGUCUGAUGAGX IGGGGAUA1256 TATCCCCC C ACCTCCCC 1980
CGAA
996 CGGGGAGGCUGAUGAGX IGGGGGAU1257 ATCCCCCC A CCTCCCCG 1981
CGAA
998 GCCGGGGACUGAUGAGX IUGGGGGG1258 CCCCCCAC C TCCCCGGC 1982
CGAA
999 GGCCGGGGCUGAUGAGX IGUGGGGG1259 CCCCCACC T CCCCGGCC 1983
CGAA
1001 GUGGCCGGCUGAUGAGX IAGGUGGG1260 CCCACCTC C CCGGCCAC 1984
CGAA
1002 GGUGGCCGCUGAUGAGX IGAGGUGG1261 CCACCTCC C CGGCCACC 1985
CGAA
1003 GGGUGGCCCUGAUGAGX 1262 CACCTCCC C GGCCACCC 1986
CGAA
IGGAGGUG
1007 GUUUGGGUCUGAUGAGX ICCGGGGA1263 TCCCCGGC C ACCCAAAC 1987
CGAA
1008 CGUUUGGGCUGAUGAGX IGCCGGGG1264 CCCCGGCC A CCCAAACG 1988
CGAA
1010 UUCGUUUGCUGAUGAGX 1265 CCGGCCAC C CAAACGAA 1989
CGAA
IUGGCCGG
1011 AUUCGUUUCUGAUGAGX IGUGGCCG1266 CGGCCACC C 1990
CGAA AAACGAAT
1012 GAUUCGUUCUGAUGAGX 1267 GGCCACCC A AACGAATC 1991
CGAA
IGGUGGCC
1021 UGGCUCCACUGAUGAGX 1268 AACGAATC C TGGAGCCA 1992
CGAA
IAUUCGUU
1022 GUGGCUCCCUGAUGAGX IGAUUCGU1269 ACGAATCC T GGAGCCAC 1993
CGAA
1028 CAUUGUGUCUGAUGAGX ICUCCAGG1270 CCTGGAGC C ACACAATG 1994
CGAA
1029 CCAUUGUGCUGAUGAGX IGCUCCAG1271 CTGGAGCC A CACAATGG 1995
CGAA
1031 UCCCAUUGCUGAUGAGX IUGGCUCC1272 GGAGCCAC A CAATGGGA 1996
CGAA
1033 UUUCCCAUCUGAUGAGX 1273 AGCCACAC A ATGGGAAA 1997
CGAA
IUGUGGCU
1045 GAACUCCCCUGAUGAGX 1274 GGAAATGC A GGGAGTTC 1998
CGAA
ICAUUUCC
1054 AUUUGGGACUGAUGAGX IAACUCCC1275 GGGAGTTC T TCCCAAAT 1999
CGAA
1057 GUGAUUUGCUGAUGAGX IAAGAACU1276 AGTTCTTC C CAAATCAC 2000
CGAA
1058 GGUGAUUU X IGAAGAAC1277 GTTCTTCC C 2001
CUGAUGAG CGAA AAATCACC
1059 UGGUGAUU X IGGAAGAA1278 TTCTTCCC A 2002
CUGAUGAG CGAA AATCACCA
1064 CCCACUGGCUGAUGAGX IAUUUGGG1279 CCCAAATC A CCAGTGGG 2003
CGAA
1066 CACCCACUCUGAUGAGX IUGAUUUG1280 CAAATCAC C AGTGGGTG 2004
CGAA
1067 UCACCCAC X 1281 AAATCACC A GTGGGTGA 2005
CUGAUGAG CGAA
IGUGAUUU
1086 UCCUCCUGCUGAUGAGX IUCUCUUC1282 GAAGAGAC C CAGGAGGA 2006
CGAA
1087 AUCCUCCUCUGAUGAGX 1283 AAGAGACC C AGGAGGAT 2007
CGAA
IGUCUCUU
1088 UAUCCUCCCUGAUGAGX IGGUCUCU1284 AGAGACCC A GGAGGATA 2008
CGAA
1102 GAUGGGGCCUGAUGAGX IUCUUUAU1285 ATAAAGAC T GCCCCATC 2009
CGAA
1105 CUUGAUGGCUGAUGAGX 1286 AAGACTGC C CCATCAAG 2010
CGAA
ICAGUCUU
1106 CCUUGAUGCUGAUGAGX IGCAGUCU1287 AGACTGCC C CATCAAGG 2011
CGAA
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1107 UCCUUGAUCUGAUGAGX IGGCAGUC1288 GACTGCCC C ATCAAGGA 2012
CGAA
1108 UUCCUUGACUGAUGAGX IGGGCAGU1289 ACTGCCCC A TCAAGGAA 2013
CGAA
1111 UUCUUCCUCUGAUGAGX IAUGGGGC1290 GCCCCATC A AGGAAGAA 2014
CGAA
1129 AUUUAAGGCUGAUGAGX ICUUCCUU1291 AAGGAAGC C CCTTAAAT 2015
CGAA
1130 CAUUUAAGCUGAUGAGX IGCUUCCU1292 AGGAAGCC C CTTAAATG 2016
CGAA
1131 GCAUUUAA X IGGCUUCC1293 GGAAGCCC C TTAAATGC 2017
CUGAUGAG CGAA
1132 GGCAUUUACUGAUGAGX IGGGCUUC1294 GAAGCCCC T TAAATGCC 2018
CGAA
1140 UAGGGUGCCUGAUGAGX ICAUUUAA1295 TTAAATGC C GCACCCTA 2019
CGAA
1143 CCGUAGGGCUGAUGAGX ICGGCAUU1296 AATGCCGC A CCCTACGG 2020
CGAA
1145 UGCCGUAGCUGAUGAGX IUGCGGCA1297 TGCCGCAC C CTACGGCA 2021
CGAA
1146 AUGCCGUACUGAUGAGX 1298 GCCGCACC C TACGGCAT 2022
CGAA
IGUGCGGC
1147 GAUGCCGUCUGAUGAGX IGGUGCGG1299 CCGCACCC T ACGGCATC 2023
CGAA
1153 GCUUUCGACUGAUGAGX ICCGUAGG1300 CCTACGGC A TCGAAAGC 2024
CGAA
1162 UUGACUCACUGAUGAGX ICUUUCGA1301 TCGAAAGC A TGAGTCAA 2025
CGAA
1169 CAGUGUCUCUGAUGAGX IACUCAUG1302 CATGAGTC A AGACACTG 2026
CGAA
1174 AACUUCAGCUGAUGAGX IUCUUGAC1303 GTCAAGAC A CTGAAGTT 2027
CGAA
1176 CUAACUUCCUGAUGAGX IUGUCUUG1304 CAAGACAC T GAAGTTAG 2028
CGAA
1208 CACCUCGACUGAUGAGX IACUUCCC1305 GGGAAGTC T TCGAGGTG 2029
CGAA
1218 GCAGCCUGCUGAUGAGX ICACCUCG1306 CGAGGTGC C CAGGCTGC 2030
CGAA .
1219 GGCAGCCUCUGAUGAGX IGCACCUC1307 GAGGTGCC C AGGCTGCC 2031
CGAA
1220 AGGCAGCCCUGAUGAGX IGGCACCU1308 AGGTGCCC A GGCTGCCT 2032
CGAA
1224 GGGGAGGCCUGAUGAGX ICCUGGGC1309 GCCCAGGC T GCCTCCCC 2033
CGAA
1227 GCUGGGGACUGAUGAGX ICAGCCUG1310 CAGGCTGC C TCCCCAGC 2034
CGAA
1228 GGCUGGGGCUGAUGAGX IGCAGCCU1311 AGGCTGCC T CCCCAGCC 2035
CGAA
1230 UUGGCUGGCUGAUGAGX IAGGCAGC1312 GCTGCCTC C CCAGCCAA 2036
CGAA
1231 UUUGGCUGCUGAUGAGX 1313 CTGCCTCC C CAGCCAAA 2037
CGAA
IGAGGCAG
1232 CUUUGGCUCUGAUGAGX IGGAGGCA1314 TGCCTCCC C AGCCAAAG 2038
CGAA
1233 CCUUUGGCCUGAUGAGX 1315 GCCTCCCC A GCCAAAGG 2039
CGAA
IGGGAGGC
1236 UCCCCUUUCUGAUGAGX ICUGGGGA1316 TCCCCAGC C 2040
CGAA AAAGGGGA
1237 CUCCCCUU X IGCUGGGG1317 CCCCAGCC A 2041
CUGAUGAG CGAA AAGGGGAG
1247 GCAGUGACCUGAUGAGX ICUCCCCU1318 AGGGGAGC C GTCACTGC 2042
CGAA
1251 UCGGGCAGCUGAUGAGX IACGGCUC1319 GAGCCGTC A CTGCCCGA 2043
CGAA
1253 UCUCGGGCCUGAUGAGX 1320 GCCGTCAC T GCCCGAGA 2044
CGAA
IUGACGGC
1256 CCUUCUCGCUGAUGAGX ICAGUGAC1321 GTCACTGC C CGAGAAGG 2045
CGAA
1257 UCCUUCUCCUGAUGAGX 1322 TCACTGCC C GAGAAGGA 2046
CGAA
IGCAGUGA
1273 CAGUGCAUCUGAUGAGX IUCCUCGU1323 ACGAGGAC C ATGCACTG 2047
CGAA
1274 UCAGUGCACUGAUGAGX IGUCCUCG1324 CGAGGACC A TGCACTGA 2048
CGAA
1278 UAACUCAGCUGAUGAGX 1325 GACCATGC A CTGAGTTA 2049
CGAA
ICAUGGUC
1280 AGUAACUCCUGAUGAGX IUGCAUGG1326 CCATGCAC T GAGTTACT 2050
CGAA
1288 GGGCUUCCCUGAUGAGX IUAACUCA1327 TGAGTTAC T GGAAGCCC 2051
CGAA
1295 CCAGGAAGCUGAUGAGX ICUUCCAG1328 CTGGAAGC C CTTCCTGG 2052
CGAA
1296 ACCAGGAA X IGCUUCCA1329 TGGAAGCC C TTCCTGGT 2053
CUGAUGAG CGAA
1297 GACCAGGACUGAUGAGX IGGCUUCC1330 GGAAGCCC T TCCTGGTC 2054
CGAA
1300 GUUGACCACUGAUGAGX IAAGGGCU1331 AGCCCTTC C TGGTCAAC 2055
CGAA
1301 UGUUGACCCUGAUGAGX IGAAGGGC1332 GCCCTTCC T 2056
CGAA GGTCAACA
1306 GCACAUGUCUGAUGAGX 1333 TCCTGGTC A ACATGTGC 2057
CGAA
IACCAGGA
1309 CACGCACACUGAUGAGX IUUGACCA1334 TGGTCAAC A TGTGCGTG 2058
CGAA
CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
198
Table 4
1320 AGGACCGUCUGAUGAGX 1335 TGCGTGGC T ACGGTCCT 2059
CGAA
ICCACGCA
1327 GGCCGUGACUGAUGAGX IACCGUAG1336 CTACGGTC C TCACGGCC 2060
CGAA
1328 CGGCCGUGCUGAUGAGX IGACCGUA1337 TACGGTCC T CACGGCCG 2061
CGAA
1330 GCCGGCCGCUGAUGAGX IAGGACCG1338 CGGTCCTC A CGGCCGGC 2062
CGAA
1335 UAAGCGCCCUGAUGAGX 1339 CTCACGGC C GGCGCTTA 2063
CGAA
ICCGUGAG
1341 CAGAGGUACUGAUGAGX ICGCCGGC1340 GCCGGCGC T TACCTCTG 2064
CGAA
1345 GUAGCAGACUGAUGAGX IUAAGCGC1341 GCGCTTAC C TCTGCTAC 2065
CGAA
1346 UGUAGCAGCUGAUGAGX IGUAAGCG1342 CGCTTACC T CTGCTACA 2066
CGAA
1348 CCUGUAGCCUGAUGAGX IAGGUAAG1343 CTTACCTC T GCTACAGG 2067
CGAA
1351 GAACCUGUCUGAUGAGX ICAGAGGU1344 ACCTCTGC T ACAGGTTC 2068
CGAA
1354 CAGGAACCCUGAUGAGX IUAGCAGA1345 TCTGCTAC A GGTTCCTG 2069
CGAA
1360 GUUGAACA X IAACCUGU1346 ACAGGTTC C TGTTCAAC 2070
CUGAUGAG CGAA
1361 UGUUGAACCUGAUGAGX IGAACCUG1347 CAGGTTCC T GTTCAACA 2071
CGAA
1366 GUUGCUGUCUGAUGAGX IAACAGGA1348 TCCTGTTC A ACAGCAAC 2072
CGAA
1369 UGUGUUGCCUGAUGAGX IUUGAACA1349 TGTTCAAC A GCAACACA 2073
CGAA
1372 CUAUGUGUCUGAUGAGX ICUGUUGA1350' TCAACAGC A ACACATAG 2074
CGAA
1375 AGGCUAUGCUGAUGAGX IUUGCUGU1351 ACAGCAAC A CATAGCCT 2075
CGAA
1377 UCAGGCUACUGAUGAGX IUGUUGCU1352 AGCAACAC A TAGCCTGA 2076
CGAA
1382 GAGGGUCACUGAUGAGX 1353 CACATAGC C TGACCCTC 2077
CGAA
ICUAUGUG
1383 GGAGGGUCCUGAUGAGX 1354 ACATAGCC T GACCCTCC 2078
CGAA
IGCUAUGU
1387 UGGAGGAGCUGAUGAGX 1355 AGCCTGAC C CTCCTCCA 2079
CGAA
IUCAGGCU
1388 GUGGAGGACUGAUGAGX 1356 GCCTGACC C TCCTCCAC 2080
CGAA
IGUCAGGC
1389 AGUGGAGGCUGAUGAGX IGGUCAGG1357 CCTGACCC T CCTCCACT 2081
CGAA
1391 GGAGUGGACUGAUGAGX IAGGGUCA1358 TGACCCTC C TCCACTCC 2082
CGAA
1392 UGGAGUGGCUGAUGAGX 1359 GACCCTCC T CCACTCCA 2083
CGAA
IGAGGGUC
1394 GGUGGAGUCUGAUGAGX IAGGAGGG1360 CCCTCCTC C ACTCCACC 2084
CGAA
1395 AGGUGGAG X IGAGGAGG1361 CCTCCTCC A CTCCACCT 2085
CUGAUGAG CGAA
1397 GGAGGUGGCUGAUGAGX IUGGAGGA1362 TCCTCCAC T CCACCTCC 2086
CGAA
1399 GUGGAGGUCUGAUGAGX IAGUGGAG1363 CTCCACTC C ACCTCCAC 2087
CGAA
1400 GGUGGAGGCUGAUGAGX IGAGUGGA1364 TCCACTCC A CCTCCACC 2088
CGAA
1402 UGGGUGGACUGAUGAGX IUGGAGUG1365 CACTCCAC C TCCACCCA 2089
CGAA
1403 GUGGGUGGCUGAUGAGX IGUGGAGU1366 ACTCCACC T CCACCCAC 2090
CGAA
1405 CAGUGGGUCUGAUGAGX IAGGUGGA1367 TCCACCTC C ACCCACTG 2091
CGAA
1406 ACAGUGGGCUGAUGAGX IGAGGUGG1368 CCACCTCC A CCCACTGT 2092
CGAA
1408 GGACAGUGCUGAUGAGX 1369 ACCTCCAC C CACTGTCC 2093
CGAA
IUGGAGGU
1409 CGGACAGUCUGAUGAGX 1370 CCTCCACC C ACTGTCCG 2094
CGAA
IGUGGAGG
1410 GCGGACAGCUGAUGAGX IGGUGGAG1371 CTCCACCC A CTGTCCGC 2095
CGAA
1412 AGGCGGACCUGAUGAGX 1372 CCACCCAC T GTCCGCCT 2096
CGAA
IUGGGUGG
1416 GCAGAGGCCUGAUGAGX IACAGUGG1373 CCACTGTC C GCCTCTGC 2097
CGAA
1419 CGGGCAGACUGAUGAGX ICGGACAG1374 CTGTCCGC C TCTGCCCG 2098
CGAA
1420 GCGGGCAGCUGAUGAGX IGCGGACA1375 TGTCCGCC T CTGCCCGC 2099
CGAA
1422 CUGCGGGCCUGAUGAGX IAGGCGGA1376 TCCGCCTC T GCCCGCAG 2100
CGAA
1425 GCUCUGCGCUGAUGAGX ICAGAGGC1377 GCCTCTGC C CGCAGAGC 2101
CGAA
1426 GGCUCUGCCUGAUGAGX IGCAGAGG1378 CCTCTGCC C GCAGAGCC 2102
CGAA
1429 GUGGGCUCCUGAUGAGX ICGGGCAG1379 CTGCCCGC A GAGCCCAC 2103
CGAA
1434 CGGGCGUGCUGAUGAGX ICUCUGCG1390 CGCAGAGC C CACGCCCG 2104
CGAA
1435 UCGGGCGUCUGAUGAGX IGCUCUGC1381 GCAGAGCC C ACGCCCGA 2105
CGAA
CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
199
Table 4
1436 GUCGGGCGCUGAUGAGX IGGCUCUG1382 CAGAGCCC A CGCCCGAC 2106
CGAA
1440 GCUAGUCGCUGAUGAGX ICGUGGGC1383 GCCCACGC C CGACTAGC 2107
CGAA
1441 UGCUAGUCCUGAUGAGX IGCGUGGG1384 CCCACGCC C GACTAGCA 2108
CGAA
1445 UGCCUGCUCUGAUGAGX IUCGGGCG1385 CGCCCGAC T AGCAGGCA 2109
CGAA
1449 GGCAUGCCCUGAUGAGX ICUAGUCG1386 CGACTAGC A GGCATGCC 2110
CGAA
1453 CCGCGGCACUGAUGAGX ICCUGCUA1387 TAGCAGGC A TGCCGCGG 2111
CGAA
1457 CCUACCGCCUGAUGAGX ICAUGCCU1388 AGGCATGC C GCGGTAGG 2112
CGAA
1473 GGUCCGGCCUGAUGAGX ICCCUUAC1389 GTAAGGGC C GCCGGACC 2113
CGAA
1476 CGCGGUCCCUGAUGAGX ICGGCCCU1390 AGGGCCGC C GGACCGCG 2114
CGAA
1481 CUCUACGCCUGAUGAGX IUCCGGCG1391 CGCCGGAC C GCGTAGAG 2115
CGAA
1493 CGGGGCCCCUGAUGAGX ICUCUCUA1392 TAGAGAGC C GGGCCCCG 2116
CGAA
1498 CCGUCCGGCUGAUGAGX ICCCGGCU1393 AGCCGGGC C CCGGACGG 2117
CGAA
1499 UCCGUCCGCUGAUGAGX IGCCCGGC1394 GC.CGGGCC C CGGACGGA 2118
CGAA
1500 GUCCGUCCCUGAUGAGX IGGCCCGG1395 CCGGGCCC C GGACGGAC 2119
CGAA
1517 UUUAGUGCCUGAUGAGX IAACCAAC1396 GTTGGTTC T GCACTAAA 2120
CGAA
1520 GGUUUUAGCUGAUGAGX ICAGAACC1397 GGTTCTGC A CTAAAACC 2121
CGAA
1522 UGGGUUUU X IUGCAGAA1398 TTCTGCAC T 2122
CUGAUGAG CGAA AAAACCCA
1528 GGAAGAUGCUGAUGAGX IUUUUAGU1399 ACTAAAAC C CATCTTCC 2123
CGAA
1529 GGGAAGAUCUGAUGAGX IGUUUUAG1400 CTAAAACC C ATCTTCCC 2124
CGAA
1530 GGGGAAGACUGAUGAGX IGGUUUUA1401 TAAAACCC A TCTTCCCC 2125
CGAA
1533 UCCGGGGACUGAUGAGX IAUGGGUU1402 AACCCATC T TCCCCGGA 2126.
CGAA
1536 ACAUCCGGCUGAUGAGX IAAGAUGG1403 CCATCTTC C CCGGATGT 2127
CGAA
1537 CACAUCCGCUGAUGAGX IGAAGAUG1404 CATCTTCC C CGGATGTG 2128
CGAA
1538 ACACAUCCCUGAUGAGX IGGAAGAU1405 ATCTTCCC C GGATGTGT 2129
CGAA
1550 GAGGGGUGCUGAUGAGX 1406 TGTGTGTC T CACCCCTC 2130
CGAA
IACACACA
1552 AUGAGGGGCUGAUGAGX IAGACACA1407 TGTGTCTC A CCCCTCAT 2131
CGAA
1554 GGAUGAGGCUGAUGAGX IUGAGACA1408 TGTCTCAC C CCTCATCC 2132
CGAA
1555 AGGAUGAGCUGAUGAGX IGUGAGAC1409 GTCTCACC C CTCATCCT 2133
CGAA
1556 AAGGAUGACUGAUGAGX IGGUGAGA1410 TCTCACCC C TCATCCTT 2134
CGAA
1557 AAAGGAUGCUGAUGAGX IGGGUGAG1411 CTCACCCC T CATCCTTT 2135
CGAA
1559 UAAAAGGACUGAUGAGX IAGGGGUG1412 CACCCCTC A TCCTTTTA 2136
CGAA
1562 AAGUAAAA X IAUGAGGG1413 CCCTCATC C TTTTACTT 2137
CUGAUGAG CGAA
1563 AAAGUAAACUGAUGAGX IGAUGAGG1414 CCTCATCC T TTTACTTT 2138
CGAA
1569 GGGCAAAACUGAUGAGX IUAAAAGG1415 CCTTTTAC T TTTTGCCC 2139
CGAA
1576 GUGGAAGGCUGAUGAGX ICAAAAAG1416 CTTTTTGC C CCTTCCAC 2140
CGAA
1577 AGUGGAAGCUGAUGAGX IGCAAAAA1417 TTTTTGCC C CTTCCACT 2141
CGAA
1578 AAGUGGAA X IGGCAAAA1418 TTTTGCCC C TTCCACTT 2142
CUGAUGAG CGAA
1579 AAAGUGGACUGAUGAGX IGGGCAAA1419 TTTGCCCC T TCCACTTT 2143
CGAA
1582 CUCAAAGU X IAAGGGGC1420 GCCCCTTC C ACTTTGAG 2144
CUGAUGAG CGAA
1583 ACUCAAAGCUGAUGAGX IGAAGGGG1421 CCCCTTCC A CTTTGAGT 2145
CGAA
1585 GUACUCAA X IUGGAAGG1422 CCTTCCAC T TTGAGTAC 2146
CUGAUGAG CGAA
1594 GUGGAUUU X IUACUCAA1423 TTGAGTAC C 2147
CUGAUGAG CGAA AAATCCAC
1595 UGUGGAW CUGAUGAGX IGUACUCA1424 TGAGTACC A 2148
CGAA AATCCACA
1600 UGGCUUGUCUGAUGAGX IAUUUGGU1425 ACCAAATC C ACAAGCCA 2149
CGAA
1601 AUGGCUUGCUGAUGAGX IGAUUUGG1426 CCAAATCC A CAAGCCAT 2150
CGAA
1603 AAAUGGCU X IUGGAUW 1427 AAATCCAC A AGCCATTT 2151
CUGAUGAG CGAA
1607 CAAAAAAUCUGAUGAGX ICUUGUGG1428 CCACAAGC C ATTTTTTG 2152
CGAA
CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
200
fable 4
1608 UCAAAAAA X IGCUUGUG1429 CACAAGCC A TTTTTTGA 2153
CUGAUGAG CGAA
1636 GCCAGCAU X IUACUCUC1430 GAGAGTAC C ATGCTGGC 2154
CUGAUGAG CGAA
1637 CGCCAGCACUGAUGAGX IGUACUCU1431 AGAGTACC A TGCTGGCG 2155
CGAA
1641 GCGCCGCCCUGAUGAGX ICAUGGUA1432 TACCATGC T GGCGGCGC 2156
CGAA
1650 CUUCCCUCCUGAUGAGX ICGCCGCC1433 GGCGGCGC A GAGGGAAG 2157
CGAA
1663 CGGGUGUACUGAUGAGX ICCCCUUC1434 GAAGGGGC C TACACCCG 2158
CGAA
1664 ACGGGUGUCUGAUGAGX IGCCCCUU1435 AAGGGGCC T ACACCCGT 2159
CGAA
1667 AAGACGGGCUGAUGAGX IUAGGCCC1436 GGGCCTAC A CCCGTCTT 2160
CGAA
1669 CCAAGACG X IUGUAGGC1437 GCCTACAC C CGTCTTGG 2161
CUGAUGAG CGAA
1670 CCCAAGACCUGAUGAGX IGUGUAGG1438 CCTACACC C GTCTTGGG 2162
CGAA
1674 GAGCCCCACUGAUGAGX IACGGGUG1439 CACCCGTC T TGGGGCTC 2163
CGAA
1681 GUGGGGCGCUGAUGAGX ICCCCAAG1440 CTTGGGGC T CGCCCCAC 2164
CGAA
1685 CUGGGUGGCUGAUGAGX ICGAGCCC1441 GGGCTCGC C CCACCCAG 2165
CGAA
1686 CCUGGGUGCUGAUGAGX 1442 GGCTCGCC C CACCCAGG 2166
CGAA
IGCGAGCC
1687 CCCUGGGUCUGAUGAGX IGGCGAGC1443 GCTCGCCC C ACCCAGGG 2167
CGAA
1688 GCCCUGGGCUGAUGAGX IGGGCGAG1444 CTCGCCCC A CCCAGGGC 2168
CGAA '
1690 GAGCCCUGCUGAUGAGX IUGGGGCG1445 CGCCCCAC C CAGGGCTC 2169
CGAA
1691 GGAGCCCUCUGAUGAGX IGUGGGGC1446 GCCCCACC C AGGGCTCC 2170
CGAA
1692 GGGAGCCCCUGAUGAGX IGGUGGGG1447 CCCCACCC A GGGCTCCC 2171
CGAA '
1697 CAGGAGGGCUGAUGAGX ICCCUGGG1448 CCCAGGGC T CCCTCCTG 2172
CGAA
1699 UCCAGGAGCUGAUGAGX IAGCCCUG1449 CAGGGCTC C CTCCTGGA 2173
CGAA
1700 CUCCAGGACUGAUGAGX IGAGCCCU1450 AGGGCTCC C TCCTGGAG 2174
CGAA
1701 GCUCCAGGCUGAUGAGX IGGAGCCC1451 GGGCTCCC T CCTGGAGC 2175
CGAA
1703 AUGCUCCACUGAUGAGX IAGGGAGC1452 GCTCCCTC C TGGAGCAT 2176
CGAA
1704 GAUGCUCCCUGAUGAGX IGAGGGAG1453 CTCCCTCC T GGAGCATC 2177
CGAA
1710 GCCUGGGACUGAUGAGX ICUCCAGG1454 CCTGGAGC A TCCCAGGC 2178
CGAA
1713 CCCGCCUGCUGAUGAGX 1455 GGAGCATC C CAGGCGGG 2179
CGAA
IAUGCUCC
1714 GCCCGCCUCUGAUGAGX IGAUGCUC1456 GAGCATCC C AGGCGGGC 2180
CGAA
1715 CGCCCGCCCUGAUGAGX IGGAUGCU1457 AGCATCCC A GGCGGGCG 2181
CGAA
1726 GUCUGGCGCUGAUGAGX ICCGCCCG1458 CGGGCGGC A CGCCAGAC 2182
CGAA
1730 GGCUGUCUCUGAUGAGX ICGUGCCG1459 CGGCACGC C AGACAGCC 2183
CGAA
1731 GGGCUGUCCUGAUGAGX IGCGUGCC1460 GGCACGCC A GACAGCCC 2184
CGAA
1735 GGGGGGGCCUGAUGAGX IUCUGGCG1461 CGCCAGAC A GCCCCCCC 2185
CGAA
1738 AAGGGGGG X ICUGUCUG1462 CAGACAGC C CCCCCCTT 2186
CUGAUGAG CGAA
1739 CAAGGGGG X IGCUGUCU1463 AGACAGCC C CCCCCTTG 2187
CUGAUGAG CGAA
1740 UCAAGGGGCUGAUGAGX IGGCUGUC1464 GACAGCCC C CCCCTTGA 2188
CGAA
1741 UUCAAGGGCUGAUGAGX IGGGCUGU1465 ACAGCCCC C CCCTTGAA 2189
CGAA
1742 AUUCAAGGCUGAUGAGX IGGGGCUG1466 CAGCCCCC C CCTTGAAT 2190
CGAA
1743 GAUUCAAGCUGAUGAGX IGGGGGCU1467 AGCCCCCC C CTTGAATC 2191
CGAA
1744 AGAUUCAACUGAUGAGX IGGGGGGC1468 GCCCCCCC C TTGAATCT 2192
CGAA
1745 CAGAUUCACUGAUGAGX IGGGGGGG1469 CCCCCCCC T TGAATCTG 2193
CGAA
1752 CUCCCUGCCUGAUGAGX IAUUCAAG1470 CTTGAATC T GCAGGGAG 2194
CGAA
1755 UUGCUCCCCUGAUGAGX ICAGAUUC1471 GAATCTGC A GGGAGCAA 2195
CGAA
1762 UGGAGAGUCUGAUGAGX ICUCCCUG1472 CAGGGAGC A ACTCTCCA 2196
CGAA
1765 GAGUGGAGCUGAUGAGX IUUGCUCC1473 GGAGCAAC T CTCCACTC 2197
CGAA
1767 UGGAGUGGCUGAUGAGX 1474 AGCAACTC T CCACTCCA 2198
CGAA
IAGUUGCU
1769 UAUGGAGUCUGAUGAGX IAGAGUUG1475 CAACTCTC C ACTCCATA 2199
CGAA
CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
201
Table 4
1770 AUAUGGAG CUGAUGAG X CGAA 1476 AACTCTCC A CTCCATAT 2200
IGAGAGUU
1772 AAAUAUGG CUGAUGAG X CGAA 1477 CTCTCCAC T CCATATTT 2201
IUGGAGAG
1774 AUAAAUAU CUGAUGAG X CGAA 1478 CTCCACTC C ATATTTAT 2202
IAGUGGAG
1775 AAUAAAUA CUGAUGAG X CGAA 1479 TCCACTCC A TATTTATT 2203
IGAGUGGA
1789 GAAAAAAU CUGAUGAG X CGAA 1480 ATTTAAAC A ATTTTTTC 2204
IUUUAAAU
1798 GCCUUUGG CUGAUGAG X CGAA 1481 ATTTTTTC C CCAAAGGC 2205
IAAAAAAU
1799 UGCCUUUG CUGAUGAG X CGAA 1482 TTTTTTCC C CAAAGGCA 2206
IGAAAAAA
1800 AUGCCUUU CUGAUGAG X CGAA 1483 TTTTTCCC C AAAGGCAT 2207
IGGAAAAA
1801 GAUGCCUU CUGAUGAG X CGAA 1484 TTTTCCCC A AAGGCATC 2208
IGGGAAAA
1807 ACUAUGGA CUGAUGAG X CGAA 1485 CCAAAGGC A TCCATAGT 2209
ICCUUUGG
1810 UGCACUAU CUGAUGAG X CGAA 1486 AAGGCATC C ATAGTGCA 2210
IAUGCCUU
1811 GUGCACUA CUGAUGAG X CGAA 1487 AGGCATCC A TAGTGCAC 2211
IGAUGCCU
1818 AAUGCUAG CUGAUGAG X CGAA 1488 CATAGTGC A CTAGCATT 2212
ICACUAUG
1820 AAAAUGCU CUGAUGAG X CGAA 1489 TAGTGCAC T AGCATTTT 2213
IUGCACUA
1824 CAAGAAAA CUGAUGAG X CGAA 1490 GCACTAGC A TTTTCTTG 2214
ICUAGUGC
1830 UUGGUUCA CUGAUGAG X CGAA 1491 GCATTTTC T TGAACCAA 2215
IAAAAUGC
1836 ACAUUAUU CUGAUGAG X CGAA 1492 TCTTGAAC C AATAATGT 2216
IUUCAAGA
1837 UACAUUAU CUGAUGAG X CGAA 1493 CTTGAACC A ATAATGTA 2217
IGUUCAAG
1864 UGCAAGGC CUGAUGAG X CGAA 1494 TTGATGTC A GCCTTGCA 2218
IACAUCAA
1867 UGAUGCAA CUGAUGAG X CGAA 1495 ATGTCAGC C TTGCATCA 2219
ICUGACAU
1868 UUGAUGCA CUGAUGAG X CGAA 1496 TGTCAGCC T TGCATCAA 2220
IGCUGACA
1872 GCCCUUGA CUGAUGAG X CGAA 1497 AGCCTTGC A TCAAGGGC 2221
ICAAGGCU
1875 AAAGCCCU CUGAUGAG X CGAA 1498 CTTGCATC A AGGGCTTT 2222
IAUGCAAG
1881 UUUGAUAA CUGAUGAG X CGAA 1499 TCAAGGGC T TTATCAAA 2223
ICCCUUGA
1887 GUACUUUU CUGAUGAG X CGAA 1500 GCTTTATC A AAAAGTAC 2224
IAUAAAGC
1896 UUUAUUAU CUGAUGAG X CGAA 1501 AAAAGTAC A ATAATAAA 2225
IUACUUUU
1907 CUACCUGA CUGAUGAG X CGAA 1502 AATAAATC C TCAGGTAG 2226
IAUUUAUU
1908 ACUACCUG CUGAUGAG X CGAA 1503 ATAAATCC T CAGGTAGT 2227
IGAUUUAU
1910 GUACUACC CUGAUGAG X CGAA 1504 AAATCCTC A GGTAGTAC 2228
IAGGAUUU
1919 CCAUUCCC CUGAUGAG X CGAA 1505 GGTAGTAC T GGGAATGG 2229
IUACUACC
1933 CAUGGCAA CUGAUGAG X CGAA 1506 TGGAAGGC T TTGCCATG 2230
ICCUUCCA
1938 AGGCCCAU CUGAUGAG X CGAA 1507 GGCTTTGC C ATGGGCCT 2231
ICAAAGCC
1939 CAGGCCCA CUGAUGAG X CGAA 1508 GCTTTGCC A TGGGCCTG 2232
IGCAAAGC
1945 ACGCAGCA CUGAUGAG X CGAA 1509 CCATGGGC C TGCTGCGT 2233
ICCCAUGG
1946 GACGCAGC CUGAUGAG X CGAA 1510 CATGGGCC T GCTGCGTC 2234
IGCCCAUG
1949 UCUGACGC CUGAUGAG X CGAA 1511 GGGCCTGC T GCGTCAGA 2235
ICAGGCCC
1955 UACUGGUC CUGAUGAG X CGAA 1512 GCTGCGTC A GACCAGTA 2236
IACGCAGC
1959 CCAGUACU CUGAUGAG X CGAA 1513 CGTCAGAC C AGTACTGG 2237
IUCUGACG
1960 CCCAGUAC CUGAUGAG X CGAA 1514 GTCAGACC A GTACTGGG 2238
IGUCUGAC
1965 UCCUUCCC CUGAUGAG X CGAA 1515 ACCAGTAC T GGGAAGGA 2239
IUACUGGU
1988 AUAACAAC CUGAUGAG X CGAA 1516 TTGTAAGC A GTTGTTAT 2240
ICUUACAA
2032 UAUAGCAU CUGAUGAG X CGAA 1517 GATAGAAC A ATGCTATA 2241
IUUCUAUC
2037 UAUAUUAU CUGAUGAG X CGAA 1518 AACAATGC T ATAATATA 2242
ICAUUGUU
2054 UACCCACG CUGAUGAG X CGAA 1519 TAATGAAC A CGTGGGTA 2243
IUUCAUUA
2076 UCACAUCA CUGAUGAG X CGAA 1520 TAAGAAAC A TGATGTGA 2244
IUUUCUUA
2091 CGGGACAA CUGAUGAG X CGAA 1521 GAGATTAC T TTGTCCCG 2245
IUAAUCUC
2097 AAUAAGCG CUGAUGAG X CGAA 1522 ACTTTGTC C CGCTTATT 2246
IACAAAGU
CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
202
Table 4
2098 GAAUAAGCCUGAUGAGX CGAA IGACAAAG1523 CTTTGTCC C GCTTATTC 2247
2101 GCAGAAUACUGAUGAGX CGAA ICGGGACA1524 TGTCCCGC T TATTCTGC 2248
2107 CAGGGAGCCUGAUGAGX CGAA IAAUAAGC1525 GCTTATTC T GCTCCCTG 2249
2110 UAACAGGGCUGAUGAGX CGAA ICAGAAUA1526 TATTCTGC T CCCTGTTA 2250
2112 GAUAACAGCUGAUGAGX CGAA IAGCAGAA1527 TTCTGCTC C CTGTTATC 2251
2113 AGAUAACACUGAUGAGX CGAA IGAGCAGA1528 TCTGCTCC C TGTTATCT 2252
2114 CAGAUAACCUGAUGAGX CGAA IGGAGCAG1529 CTGCTCCC T GTTATCTG 2253
2121 GAUCUAGCCUGAUGAGX CGAA IAUAACAG1530 CTGTTATC T GCTAGATC 2254
2124 CUAGAUCUCUGAUGAGX CGAA ICAGAUAA1531 TTATCTGC T AGATCTAG 2255
2130 UGAGAACUCUGAUGAGX CGAA IAUCUAGC1532 GCTAGATC T AGTTCTCA 2256
2136 AGUGAUUGCUGAUGAGX CGAA IAACUAGA1533 TCTAGTTC T CAATCACT 2257
2138 GCAGUGAUCUGAUGAGX CGAA IAGAACUA1534 TAGTTCTC A ATCACTGC 2258
2142 GGGAGCAGCUGAUGAGX CGAA IAUUGAGA1535 TCTCAATC A CTGCTCCC 2259
2144 GGGGGAGCCUGAUGAGX CGAA IUGAUUGA1536 TCAATCAC T GCTCCCCC 2260
2147 CACGGGGGCUGAUGAGX CGAA ICAGUGAU1537 ATCACTGC T CCCCCGTG 2261
2149 CACACGGGCUGAUGAGX CGAA IAGCAGUG1538 CACTGCTC C CCCGTGTG 2262
2150 ACACACGGCUGAUGAGX CGAA IGAGCAGU1539 ACTGCTCC C CCGTGTGT 2263
2151 UACACACGCUGAUGAGX CGAA IGGAGCAG1540 CTGCTCCC C CGTGTGTA 2264
2152 AUACACACCUGAUGAGX CGAA IGGGAGCA1541 TGCTCCCC C GTGTGTAT 2265
2169 ACCUUACACUGAUGAGX CGAA ICAUUCUA1542 TAGAATGC A TGTAAGGT 2266
2179 ACACAAGACUGAUGAGX CGAA IACCUUAC1543 GTAAGGTC T TCTTGTGT 2267
2182 AGGACACACUGAUGAGX CGAA IAAGACCU1544 AGGTCTTC T TGTGTCCT 2268
2189 UUUCAUCACUGAUGAGX CGAA IACACAAG1545 CTTGTGTC C TGATGAAA 2269
2190 UUUUCAUCCUGAUGAGX CGAA IGACACAA1546 TTGTGTCC T GATGAAAA 2270
2207 UCAUUUCACUGAUGAGX CGAA ICACAUAU1547 ATATGTGC T TGAAATGA 2271
2221 GAGAUCAACUGAUGAGX CGAA IUUUCUCA1548 TGAGAAAC T TTGATCTC 2272
2228 GUAAGCAGCUGAUGAGX CGAA IAUCAAAG1549 CTTTGATC T CTGCTTAC 2273
2230 UAGUAAGCCUGAUGAGX CGAA IAGAUCAA1550 TTGATCTC T GCTTACTA 2274
2233 CAUUAGUACUGAUGAGX CGAA ICAGAGAU1551 ATCTCTGC T TACTAATG 2275
2237 GGCACAUU X CGAA IUAAGCAG1552 CTGCTTAC T 2276
CUGAUGAG AATGTGCC
2245 GGACAUGGCUGAUGAGX CGAA ICACAUUA1553 TAATGTGC C CCATGTCC 2277
2246 UGGACAUGCUGAUGAGX CGAA IGCACAUU1554 AATGTGCC C CATGTCCA 2278
2247 UUGGACAUCUGAUGAGX CGAA IGGCACAU1555 ATGTGCCC C ATGTCCAA 2279
2248 CUUGGACACUGAUGAGX CGAA IGGGCACA1556 TGTGCCCC A TGTCCAAG 2280
2253 UUGGACUU X CGAA IACAUGGG1557 CCCATGTC C 2281
CUGAUGAG AAGTCCAA
2254 GUUGGACUCUGAUGAGX CGAA IGACAUGG1558 CCATGTCC A AGTCCAAC 2282
2259 GGCAGGUU X CGAA IACUUGGA1559 TCCAAGTC C AACCTGCC 2283
CUGAUGAG
2260 AGGCAGGUCUGAUGAGX CGAA IGACUUGG1560 CCAAGTCC A ACCTGCCT 2284
2263 CACAGGCACUGAUGAGX CGAA IUUGGACU1561 AGTCCAAC C TGCCTGTG 2285
2264 GCACAGGCCUGAUGAGX CGAA IGUUGGAC1562 GTCCAACC T GCCTGTGC 2286
2267 CAUGCACACUGAUGAGX CGAA ICAGGUUG1563 CAACCTGC C TGTGCATG 2287
2268 UCAUGCACCUGAUGAGX CGAA IGCAGGUU1564 AACCTGCC T GTGCATGA 2288
2273 UCAGGUCACUGAUGAGX CGAA ICACAGGC1565 GCCTGTGC A TGACCTGA 2289
2278 AAUGAUCACUGAUGAGX CGAA IUCAUGCA1566 TGCATGAC C TGATCATT 2290
2279 UAAUGAUCCUGAUGAGX CGAA IGUCAUGC1567 GCATGACC T GATCATTA 2291
2284 CCAUGUAA X CGAA IAUCAGGU1568 ACCTGATC A TTACATGG 2292
CUGAUGAG
2289 CACAGCCACUGAUGAGX CGAA IUAAUGAU1569 ATCATTAC A TGGCTGTG 2293
CA 02403243 2002-02-21
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203
Table 4
2294 GGAACCACCUGAUGAGX ICCAUGUA1570 TACATGGC T GTGGTTCC 2294
CGAA
2302 CAGGCUUACUGAUGAGX IAACCACA1571 TGTGGTTC C TAAGCCTG 2295
CGAA
2303 ACAGGCUU X IGAACCAC1572 GTGGTTCC T AAGCCTGT 2296
CUGAUGAG CGAA
2308 CAGCAACACUGAUGAGX ICUUAGGA1573 TCCTAAGC C TGTTGCTG 2297
CGAA
2309 UCAGCAACCUGAUGAGX IGCUUAGG1574 CCTAAGCC T GTTGCTGA 2298
CGAA
2315 AUGACUUCCUGAUGAGX ICAACAGG1575 CCTGTTGC T GAAGTCAT 2299
CGAA
2322 AGCGACAA X IACUUCAG1576 CTGAAGTC A TTGTCGCT 2300
CUGAUGAG CGAA
2330 UAUUGCUGCUGAUGAGX ICGACAAU1577 ATTGTCGC T CAGCAATA 2301
CGAA
2332 CCUAUUGCCUGAUGAGX IAGCGACA1578 TGTCGCTC A GCAATAGG 2302
CGAA
2335 CACCCUAUCUGAUGAGX ICUGAGCG1579 CGCTCAGC A ATAGGGTG 2303
CGAA
2345 UGGAAAACCUGAUGAGX ICACCCUA1580 TAGGGTGC A GTTTTCCA 2304
CGAA
2352 CUAUUCCUCUGAUGAGX IAAAACUG1581 CAGTTTTC C AGGAATAG 2305
CGAA
2353 CCUAUUCCCUGAUGAGX IGAAAACU1582 AGTTTTCC A GGAATAGG 2306
CGAA
2363 UAGGCAAA X ICCUAWC 1583 GAATAGGC A TTTGCCTA 2307
CUGAUGAG CGAA
2369 AGGAAUUACUGAUGAGX ICAAAUGC1584 GCATTTGC C TAATTCCT 2308
CGAA
2370 CAGGAAUU X IGCAAAUG1585 CATTTGCC T 2309
CUGAUGAG CGAA AATTCCTG
2376 UCAUGCCACUGAUGAGX IAAUUAGG1586 CCTAATTC C TGGCATGA 2310
CGAA
2377 GUCAUGCCCUGAUGAGX IGAAUUAG1587 CTAATTCC T GGCATGAC 2311
CGAA
2381 GAGUGUCACUGAUGAGX ICCAGGAA1588 TTCCTGGC A TGACACTC 2312
CGAA
2386 CACUAGAGCUGAUGAGX IUCAUGCC1589 GGCATGAC A CTCTAGTG 2313
CGAA
2388 GUCACUAGCUGAUGAGX IUGUCAUG1590 CATGACAC T CTAGTGAC 2314
CGAA
2390 AAGUCACUCUGAUGAGX IAGUGUCA1591 TGACACTC T AGTGACTT 2315
CGAA
2397 CACCAGGACUGAUGAGX IUCACUAG1592 CTAGTGAC T TCCTGGTG 2316
CGAA
2400 CCUCACCACUGAUGAGX IAAGUCAC1593 GTGACTTC C TGGTGAGG 2317
CGAA
2401 GCCUCACCCUGAUGAGX IGAAGUCA1594 TGACTTCC T GGTGAGGC 2318
CGAA
2410 ACAGGCUGCUGAUGAGX ICCUCACC1595 GGTGAGGC C CAGCCTGT 2319
CGAA
2411 GACAGGCUCUGAUGAGX IGCCUCAC1596 GTGAGGCC C AGCCTGTC 2320
CGAA
2412 GGACAGGCCUGAUGAGX IGGCCUCA1597 TGAGGCCC A GCCTGTCC 2321
CGAA
2415 CCAGGACACUGAUGAGX ICUGGGCC1598 GGCCCAGC C TGTCCTGG 2322
CGAA
2416 ACCAGGACCUGAUGAGX IGCUGGGC1599 GCCCAGCC T GTCCTGGT 2323
CGAA
2420 CUGUACCACUGAUGAGX IACAGGCU1600 AGCCTGTC C TGGTACAG 2324
CGAA
2421 GCUGUACCCUGAUGAGX IGACAGGC1601 GCCTGTCC T GGTACAGC 2325
CGAA
2427 GACCCUGCCUGAUGAGX IUACCAGG1602 CCTGGTAC A GCAGGGTC 2326
CGAA
2430 CAAGACCCCUGAUGAGX ICUGUACC1603 GGTACAGC A GGGTCTTG 2327
CGAA
2436 UUACAGCACUGAUGAGX IACCCUGC1604 GCAGGGTC T TGCTGTAA 2328
CGAA
2440 UGAGUUACCUGAUGAGX ICAAGACC1605 GGTCTTGC T GTAACTCA 2329
CGAA
2446 AAUGUCUGCUGAUGAGX IUUACAGC1606 GCTGTAAC T CAGACATT 2330
CGAA
2448 GGAAUGUCCUGAUGAGX IAGUUACA1607 TGTAACTC A GACATTCC 2331
CGAA
2452 CCUUGGAA X IUCUGAGU1608 ACTCAGAC A TTCCAAGG 2332
CUGAUGAG CGAA
2456 AUACCCUU X IAAUGUCU1609 AGACATTC C 2333
CUGAUGAG CGAA AAGGGTAT
245.7 CAUACCCUCUGAUGAGX IGAAUGUC1610 GACATTCC A AGGGTATG 2334
CGAA
2472 GUGAAUAUCUGAUGAGX ICUUCCCA1611 TGGGAAGC C ATATT'CAC 2335
CGAA
2473 UGUGAAUACUGAUGAGX IGCUUCCC1612 GGGAAGCC A TATTCACA 2336
CGAA
2479 GUGAGGUGCUGAUGAGX IAAUAUGG1613 CCATATTC A CACCTCAC 2337
CGAA
2481 GCGUGAGGCUGAUGAGX IUGAAUAU1614 ATATTCAC A CCTCACGC 2338
CGAA
2483 GAGCGUGACUGAUGAGX IUGUGAAU1615 ATTCACAC C TCACGCTC 2339
CGAA
2484 AGAGCGUGCUGAUGAGX IGUGUGAA1616 TTCACACC T CACGCTCT 2340
CGAA
CA 02403243 2002-02-21
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204
Table 4
2486 CCAGAGCGCUGAUGAGX IAGGUGUG1617 CACACCTC A CGCTCTGG 2341
CGAA
2490 AUGUCCAGCUGAUGAGX ICGUGAGG1618 CCTCACGC T CTGGACAT 2342
CGAA
2492 UCAUGUCCCUGAUGAGX IAGCGUGA1619 TCACGCTC T GGACATGA 2343
CGAA
2497 CUAAAUCACUGAUGAGX IUCCAGAG1620 CTCTGGAC A TGATTTAG 2344
CGAA
2512 GGUGUCCCCUGAUGAGX ICUUCCCU1621 AGGGAAGC A GGGACACC 2345
CGAA
2518 GCGGGGGGCUGAUGAGX IUCCCUGC1622 GCAGGGAC A CCCCCCGC 2346
CGAA
2520 GGGCGGGGCUGAUGAGX IUGUCCCU1623 AGGGACAC C CCCCGCCC 2347
CGAA
2521 GGGGCGGGCUGAUGAGX IGUGUCCC1624 GGGACACC C CCCGCCCC 2348
CGAA
2522 GGGGGCGGCUGAUGAGX IGGUGUCC1625 GGACACCC C CCGCCCCC 2349
CGAA
2523 GGGGGGCGCUGAUGAGX IGGGUGUC1626 GACACCCC C CGCCCCCC 2350
CGAA
2524 UGGGGGGCCUGAUGAGX IGGGGUGU1627 ACACCCCC C GCCCCCCA 2351
CGAA
2527 AGGUGGGGCUGAUGAGX ICGGGGGG1628 CCCCCCGC C CCCCACCT 2352
CGAA
2528 AAGGUGGGCUGAUGAGX IGCGGGGG1629 CCCCCGCC C CCCACCTT 2353
CGAA
2529 AAAGGUGGCUGAUGAGX IGGCGGGG1630 CCCCGCCC C CCACCTTT 2354
CGAA
2530 CAAAGGUGCUGAUGAGX IGGGCGGG1631 CCCGCCCC C CACCTTTG 2355
CGAA
2531 CCAAAGGUCUGAUGAGX IGGGGCGG1632 CCGCCCCC C ACCTTTGG 2356
CGAA
2532 CCCAAAGGCUGAUGAGX IGGGGGCG1633 CGCCCCCC A CCTTTGGG 2357
CGAA
2534 AUCCCAAACUGAUGAGX IUGGGGGG1634 CCCCCCAC C TTTGGGAT 2358
CGAA
2535 GAUCCCAA X IGUGGGGG1635 CCCCCACC T TTGGGATC 2359
CUGAUGAG CGAA
2544 GCGGAGGCCUGAUGAGX IAUCCCAA1636 TTGGGATC A GCCTCCGC 2360
CGAA
2547 AUGGCGGACUGAUGAGX ICUGAUCC1637 GGATCAGC C TCCGCCAT 2361
CGAA
2548 AAUGGCGGCUGAUGAGX IGCUGAUC1638 GATCAGCC T CCGCCATT 2362
CGAA
2550 GGAAUGGCCUGAUGAGX IAGGCUGA1639 TCAGCCTC C GCCATTCC 2363
CGAA
2553 CUUGGAAUCUGAUGAGX ICGGAGGC1640 GCCTCCGC C ATTCCAAG 2364
CGAA
2554 ACUUGGAACUGAUGAGX IGCGGAGG1641 CCTCCGCC A TTCCAAGT 2365
CGAA
2558 GUCGACUU X IAAUGGCG1642 CGCCATTC C 2366
CUGAUGAG CGAA AAGTCGAC
2559 UGUCGACUCUGAUGAGX IGAAUGGC1643 GCCATTCC A AGTCGACA 2367
CGAA
2567 AAGAAGAGCUGAUGAGX IUCGACUU1644 AAGTCGAC A CTCTTCTT 2368
CGAA
2569 UCAAGAAGCUGAUGAGX IUGUCGAC1645 GTCGACAC T CTTCTTGA 2369
CGAA
2571 GCUCAAGACUGAUGAGX IAGUGUCG1646 CGACACTC T TCTTGAGC 2370
CGAA
2574 UCUGCUCACUGAUGAGX IAAGAGUG1647 CACTCTTC T TGAGCAGA 2371
CGAA
2580 UCACGGUCCUGAUGAGX ICUCAAGA1648 TCTTGAGC A GACCGTGA 2372
CGAA
2584 CAAAUCACCUGAUGAGX IUCUGCUC1649 GAGCAGAC C GTGATTTG 2373
CGAA
2603 CCAGCAGGCUGAUGAGX ICCUCUCU1650 AGAGAGGC A CCTGCTGG 2374
CGAA
2605 UUCCAGCACUGAUGAGX IUGCCUCU1651 AGAGGCAC C TGCTGGAA 2375
CGAA
2606 UUUCCAGCCUGAUGAGX IGUGCCUC1652 GAGGCACC T GCTGGAAA 2376
CGAA
2609 UGGUUUCCCUGAUGAGX ICAGGUGC1653 GCACCTGC T GGAAACCA 2377
CGAA
2616 AGAAGUGUCUGAUGAGX IUUUCCAG1654 CTGGAAAC C ACACTTCT 2378
CGAA
2617 AAGAAGUGCUGAUGAGX IGUWCCA 1655 TGGAAACC A CACTTCTT 2379
CGAA
2619 UCAAGAAGCUGAUGAGX IUGGUUUC1656 GAAACCAC A CTTCTTGA 2380
CGAA
2621 UUUCAAGACUGAUGAGX IUGUGGUU1657 AACCACAC T TCTTGAAA 2381
CGAA
2624 CUGUUUCACUGAUGAGX IAAGUGUG1658 CACACTTC T TGAAACAG 2382
CGAA
2631 ACCCAGGCCUGAUGAGX IUUUCAAG1659 CTTGAAAC A GCCTGGGT 2383
CGAA
2634 GUCACCCACUGAUGAGX ICUGUUUC1660 GAAACAGC C TGGGTGAC 2384
CGAA
2635 CGUCACCCCUGAUGAGX IGCUGUUU1661 AAACAGCC T GGGTGACG 2385
CGAA
2647 UGCCUAAA X IACCGUCA1662 TGACGGTC C TTTAGGCA 2386
CUGAUGAG CGAA
2648 CUGCCUAA X IGACCGUC1663 GACGGTCC T TTAGGCAG 2387
CUGAUGAG CGAA
CA 02403243 2002-02-21
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205
Table 4
2655 CGGCAGGCCUGAUGAGX ICCUAAAG1664 CTTTAGGC A GCCTGCCG 2388
CGAA
2658 CGGCGGCACUGAUGAGX ICUGCCUA1665 TAGGCAGC C TGCCGCCG 2389
CGAA
2659 ACGGCGGCCUGAUGAGX IGCUGCCU1666 AGGCAGCC T GCCGCCGT 2390
CGAA
2662 GAGACGGCCUGAUGAGX ICAGGCUG1667 CAGCCTGC C GCCGTCTC 2391
CGAA
2665 ACAGAGACCUGAUGAGX ICGGCAGG1668 CCTGCCGC C GTCTCTGT 2392
CGAA
2669 CGGGACAGCUGAUGAGX IACGGCGG1669 CCGCCGTC T CTGTCCCG 2393
CGAA
2671 ACCGGGACCUGAUGAGX IAGACGGC1670 GCCGTCTC T GTCCCGGT 2394
CGAA
2675 GUGAACCGCUGAUGAGX IACAGAGA1671 TCTCTGTC C CGGTTCAC 2395
CGAA
2676 GGUGAACCCUGAUGAGX IGACAGAG1672 CTCTGTCC C GGTTCACC 2396
CGAA
2682 CGGCAAGGCUGAUGAGX IAACCGGG1673 CCCGGTTC A CCTTGCCG 2397
CGAA
2684 CUCGGCAA X IUGAACCG1674 CGGTTCAC C TTGCCGAG 2398
CUGAUGAG CGAA
2685 UCUCGGCACUGAUGAGX IGUGAACC1675 GGTTCACC T TGCCGAGA 2399
CGAA
2689 CCUCUCUCCUGAUGAGX ICAAGGUG1676 CACCTTGC C GAGAGAGG 2400
CGAA
2704 GGUGGGGCCUGAUGAGX IACGCGCC1677 GGCGCGTC T GCCCCACC 2401
CGAA
2707 GAGGGUGGCUGAUGAGX ICAGACGC1678 GCGTCTGC C CCACCCTC 2402
CGAA
2708 UGAGGGUGCUGAUGAGX IGCAGACG1679 CGTCTGCC C CACCCTCA 2403
CGAA
2709 UUGAGGGUCUGAUGAGX IGGCAGAC1680 GTCTGCCC C ACCCTCAA 2404
CGAA
2710 UUUGAGGGCUGAUGAGX IGGGCAGA1681 TCTGCCCC A CCCTCAAA 2405
CGAA
2712 GGUUUGAGCUGAUGAGX IUGGGGCA1682 TGCCCCAC C CTCAAACC 2406
CGAA
2713 GGGUUUGACUGAUGAGX IGUGGGGC1683 GCCCCACC C TCAAACCC 2407
CGAA
2714 AGGGUUUGCUGAUGAGX IGGUGGGG1684 CCCCACCC T CAAACCCT 2408
CGAA
2716 ACAGGGUU X IAGGGUGG1685 CCACCCTC A AACCCTGT 2409
CUGAUGAG CGAA
2720 CCCCACAGCUGAUGAGX IUUUGAGG1686 CCTCAAAC C CTGTGGGG 2410
CGAA
2721 GCCCCACACUGAUGAGX IGUUUGAG1687 CTCAAACC C TGTGGGGC 2411
CGAA
2722 GGCCCCACCUGAUGAGX IGGUUUGA1688 TCAAACCC T GTGGGGCC 2412
CGAA
2730 CACCAUCACUGAUGAGX ICCCCACA1689 TGTGGGGC C TGATGGTG 2413
CGAA
2731 GCACCAUCCUGAUGAGX IGCCCCAC1690 GTGGGGCC T GATGGTGC 2414
CGAA
2740 GAGUCGUGCUGAUGAGX ICACCAUC1691 GATGGTGC T CACGACTC 2415
CGAA
2742 AAGAGUCGCUGAUGAGX IAGCACCA1692 TGGTGCTC A CGACTCTT 2416
CGAA
2747 GCAGGAAGCUGAUGAGX IUCGUGAG1693 CTCACGAC T CTTCCTGC 2417
CGAA
2749 UUGCAGGACUGAUGAGX IAGUCGUG1694 CACGACTC T TCCTGCAA 2418
CGAA
2752 CCUUUGCACUGAUGAGX IAAGAGUC1695 GACTCTTC C TGCAAAGG 2419
CGAA
2753 CCCUUUGCCUGAUGAGX IGAAGAGU1696 ACTCTTCC T GCAAAGGG 2420
CGAA
2756 GUUCCCUU X ICAGGAAG1697 CTTCCTGC A AAGGGAAC 2421
CUGAUGAG CGAA
2765 AGGUCUUCCUGAUGAGX IUUCCCUU1698 AAGGGAAC T GAAGACCT 2422
CGAA
2772 AAUGUGGACUGAUGAGX IUCUUCAG1699 CTGAAGAC C TCCACATT 2423
CGAA
2773 UAAUGUGGCUGAUGAGX IGUCUUCA1700 TGAAGACC T CCACATTA 2424
CGAA
2775 CUUAAUGUCUGAUGAGX IAGGUCUU1701 AAGACCTC C ACATTAAG 2425
CGAA
2776 ACUUAAUGCUGAUGAGX IGAGGUCU1702 AGACCTCC A CATTAAGT 2426
CGAA
2778 CCACUUAA X IUGGAGGU1703 ACCTCCAC A TTAAGTGG 2427
CUGAUGAG CGAA
2788 UGUUAAAA X ICCACUUA1704 TAAGTGGC T TTTTAACA 2428
CUGAUGAG CGAA
2796 GUUUUUCACUGAUGAGX IUUAAAAA1705 TTTTTAAC A TGAAAAAC 2429
CGAA
2805 AGCUGCCGCUGAUGAGX IUUUUUCA1706 TGAAAAAC A CGGCAGCT 2430
CGAA
2810 GCUACAGCCUGAUGAGX ICCGUGUU1707 AACACGGC A GCTGTAGC 2431
CGAA
2813 GGAGCUACCUGAUGAGX ICUGCCGU1708 ACGGCAGC T GTAGCTCC 2432
CGAA
2819 AGCUCGGG X ICUACAGC1709 GCTGTAGC T CCCGAGCT 2433'
CUGAUGAG CGAA
2821 GUAGCUCGCUGAUGAGX IAGCUACA1710 TGTAGCTC C CGAGCTAC 2434
CGAA
CA 02403243 2002-02-21
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206
Table 4
2822 AGUAGCUCCUGAUGAGX IGAGCUAC1711 GTAGCTCC C GAGCTACT 2435
CGAA
2827 AAGAGAGUCUGAUGAGX ICUCGGGA1712 TCCCGAGC T ACTCTCTT 2436
CGAA
2830 GGCAAGAGCUGAUGAGX IUAGCUCG1713 CGAGCTAC T CTCTTGCC 2437
CGAA
2832 CUGGCAAGCUGAUGAGX IAGUAGCU1714 AGCTACTC T CTTGCCAG 2438
CGAA
2834 UGCUGGCACUGAUGAGX IAGAGUAG1715 CTACTCTC T TGCCAGCA 2439
CGAA
2838 AAAAUGCUCUGAUGAGX ICAAGAGA1716 TCTCTTGC C AGCATTTT 2440
CGAA
2839 GAAAAUGCCUGAUGAGX IGCAAGAG1717 CTCTTGCC A GCATTTTC 2441
CGAA
2842 UGUGAAAACUGAUGAGX ICUGGCAA1718 TTGCCAGC A TTTTCACA 2442
CGAA
2848 GCAAAAUGCUGAUGAGX 1719 GCATTTTC A CATTTTGC 2443
CGAA
IAAAAUGC
2850 AGGCAAAA X IUGAAAAU1720 ATTTTCAC A TTTTGCCT 2444
CUGAUGAG CGAA
2857 ACGAGAAA X 1721 CATTTTGC C TTTCTCGT 2445
CUGAUGAG CGAA
ICAAAAUG
2858 CACGAGAA X 1722 ATTTTGCC T TTCTCGTG 2446
CUGAUGAG CGAA
IGCAAAAU
2862 CUACCACGCUGAUGAGX 1723 TGCCTTTC T CGTGGTAG 2447
CGAA
IAAAGGCA
2875 UCUGUACUCUGAUGAGX 1724 GTAGAAGC C AGTACAGA 2448
CGAA
ICUUCUAC
2876 CUCUGUACCUGAUGAGX IGCUUCUA1725 TAGAAGCC A GTACAGAG 2449
CGAA
2881 AAUUUCUCCUGAUGAGX IUACUGGC1726 GCCAGTAC A GAGAAATT 2450
CGAA
2891 CCCACCACCUGAUGAGX IAAUUUCU1727 AGAAATTC T GTGGTGGG 2451
CGAA
2903 ACCUCGAA X IUUCCCAC1728 GTGGGAAC A TTCGAGGT 2452
CUGAUGAG CGAA
2915 CUGCAGGGCUGAUGAGX 1729 GAGGTGTC A CCCTGCAG 2453
CGAA
IACACCUC
2917 CUCUGCAGCUGAUGAGX IUGACACC1730 GGTGTCAC C CTGCAGAG 2454
CGAA
2918 GCUCUGCACUGAUGAGX IGUGACAC1731 GTGTCACC C TGCAGAGC 2455
CGAA
2919 AGCUCUGCCUGAUGAGX 1732 TGTCACCC T GCAGAGCT 2456
CGAA
IGGUGACA
2922 CAUAGCUCCUGAUGAGX ICAGGGUG1733 CACCCTGC A GAGCTATG. 2457
CGAA
2927 CUCACCAUCUGAUGAGX ICUCUGCA1734 TGCAGAGC T ATGGTGAG 2458
CGAA
2949 GGCACCUACUGAUGAGX ICCUUAUC1735 GATAAGGC T TAGGTGCC 2459
CGAA
2957 UACAGCCUCUGAUGAGX ICACCUAA1736 TTAGGTGC C AGGCTGTA 2460
CGAA
2958 UUACAGCCCUGAUGAGX IGCACCUA1737 TAGGTGCC A GGCTGTAA 2461
CGAA
2962 AUGCUUACCUGAUGAGX ICCUGGCA1738 TGCCAGGC T GTAAGCAT 2462
CGAA
2969 GCUCAGAA X ICUUACAG1739 CTGTAAGC A TTCTGAGC 2463
CUGAUGAG CGAA
2973 GCCAGCUCCUGAUGAGX IAAUGCUU1740 AAGCATTC T GAGCTGGC 2464
CGAA
2978 AACAAGCCCUGAUGAGX ICUCAGAA1741 TTCTGAGC T GGCTTGTT 2465
CGAA
2982 AAACAACACUGAUGAGX 1742 GAGCTGGC T TGTTGTTT 2466
CGAA
ICCAGCUC
2998 CAUAUACACUGAUGAGX 1743 TTTAAGTC C TGTATATG 2467
CGAA
IACUUAAA
2999 ACAUAUACCUGAUGAGX 1744 TTAAGTCC T GTATATGT 2468
CGAA
IGACUUAA
3040 UUUUGAAA X ICUACUAU1745 ATAGTAGC A TTTCAAAA 2469
CUGAUGAG CGAA
3045 GUCCAUUU X 1746 AGCATTTC A 2470
CUGAUGAG CGAA AAATGGAC
IAAAUGCU
3058 GUUAAACCCUGAUGAGX IUACGUCC1747 GGACGTAC T GGTTTAAC 2471
CGAA
3067 GGAUAGGACUGAUGAGX IUUAAACC1748 GGTTTAAC C TCCTATCC 2472
CGAA
3068 AGGAUAGGCUGAUGAGX IGUUAAAC1749 GTTTAACC T CCTATCCT 2473
CGAA
3070 CAAGGAUACUGAUGAGX IAGGUUAA1750 TTAACCTC C TATCCTTG 2474
CGAA
3071 CCAAGGAUCUGAUGAGX 1751 TAACCTCC T ATCCTTGG 2475
CGAA
IGAGGUUA
3075 CUCUCCAA X 1752 CTCCTATC C TTGGAGAG 2476
CUGAUGAG CGAA
IAUAGGAG
3076 GCUCUCCACUGAUGAGX IGAUAGGA1753 TCCTATCC T TGGAGAGC 2477
CGAA
3085 GAGCCAGCCUGAUGAGX 1754 TGGAGAGC A GCTGGCTC 2478
CGAA
ICUCUCCA
3088 GGAGAGCCCUGAUGAGX ICUGCUCU1755 AGAGCAGC T 2479
CGAA GGCTCTCC
3092 AGGUGGAGCUGAUGAGX ICCAGCUG1756 CAGCTGGC T CTCCACCT 2480
CGAA
3094 CAAGGUGGCUGAUGAGX IAGCCAGC1757 GCTGGCTC T CCACCTTG 2481
CGAA
CA 02403243 2002-02-21
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207
Table 4
3096 AACAAGGUCUGAUGAGXCGAAIAGAGCCA1758 TGGCTCTC C ACCTTGTT 2482
3097 UAACAAGGCUGAUGAGXCGAAIGAGAGCC1759 GGCTCTCC A CCTTGTTA 2483
3099 UGUAACAA XCGAAIUGGAGAG1760 CTCTCCAC C TTGTTACA 2484
CUGAUGAG
3100 GUGUAACACUGAUGAGXCGAAIGUGGAGA1761 TCTCCACC T TGTTACAC 2485
3107 ACAUAAUGCUGAUGAGXCGAAIUAACAAG1762 CTTGTTAC A CATTATGT 2486
3109 UAACAUAA XCGAAIUGUAACA1763 TGTTACAC A TTATGTTA 2487
CUGAUGAG
3132 AGCAGAGCCUGAUGAGXCGAAICUCGCUA1764 TAGCGAGC T GCTCTGCT 2488
3135 CAUAGCAGCUGAUGAGXCGAAICAGCUCG1765 CGAGCTGC T CTGCTATG 2489
3137 GACAUAGCCUGAUGAGXCGAAIAGCAGCU1766 AGCTGCTC T GCTATGTC 2490
3140 AAGGACAUCUGAUGAGXCGAAICAGAGCA1767 TGCTCTGC T ATGTCCTT 2491
3146 UGGCUUAA XCGAAIACAUAGC1768 GCTATGTC C TTAAGCCA 2492
CUGAUGAG
3147 UUGGCUUACUGAUGAGXCGAAIGACAUAG1769 CTATGTCC T TAAGCCAA 2493
3153 UAAAUAUUCUGAUGAGXCGAAICUUAAGG1770 CCTTAAGC C 2494
AATATTTA
3154 GUAAAUAUCUGAUGAGXCGAAIGCUUAAG1771 CTTAAGCC A ATATTTAC 2495
3163 ACCUGAUGCUGAUGAGXCGAAIUAAAUAU1772 ATATTTAC T CATCAGGT 2496
3165 UGACCUGACUGAUGAGXCGAAIAGUAAAU1773 ATTTACTC A TCAGGTCA 2497
3168 UAAUGACCCUGAUGAGXCGAAIAUGAGUA1774 TACTCATC A GGTCATTA 2498
3173 AAAAAUAACUGAUGAGXCGAAIACCUGAU1775 ATCAGGTC A TTATTTTT 2499
3185 AUGGCCAUCUGAUGAGXCGAAIUAAAAAA1776 TTTTTTAC A ATGGCCAT 2500
3191 UAUUCCAUCUGAUGAGXCGAAICCAUUGU1777 ACAATGGC C ATGGAATA 2501
3192 UUAUUCCACUGAUGAGXCGAAIGCCAUUG1778 CAATGGCC A TGGAATAA 2502
-
3203 GUAAAAAUCUGAUGAGXCGAAIUUUAUUC1779 GAATAAAC C ATTTTTAC 2503
3204 UGUAAAAA XCGAAIGUUUAUU1780 AATAAACC A TTTTTACA 2504
CUGAUGAG
CA 02403243 2002-02-21
WO 01/16312 PCT/US00/23998
208
N O r-IN M CWf1~Of~0001O r1N M C~1f1l0l~c001O ~-IN M VWf1\Ol~
O I~l~f~l~f~l~I~L~hI~W COODN ODW N ODODc00101Q1O~O~O~O~O~
Q,~D~O~O10~OlDlDtD~D~D~D~O~O1D~Ol0lO~Dl0l0~Ol0~D~O~O~O~O~O
~
y N N N N N N N N NN N N N N N N N NN N N N N N N N NN
C7C~a C7C7C7C~U Ua F F H U aU F U H U F a Ua
H U F a F U FH a a C7 ~ H ~ t7a
~ ~ F a
C7~ C~U U ~ a C7F U F a F C7U C7U U U C U C ~ ~
a C7a U U U U U~ U U F H U C7F FC7a F 7 ~ F 7
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Table 9
Table 9: Human, methionine aminopeptidase type 2 (Met AP-2) Hammerhead
Ribozyme and Target Sequence
Nt. Riboryme Sequence Seq Substrate Sequence Seq
osition ID 1D
nos. nos.
9 CCGAGAGA CUGAUGAG X CGAA ACGAGGGA1 TCCCTCGT C TCTCTCGG 413
11 GCCCGAGA CUGAUGAG X CGAA AGACGAGG2 CCTCGTCT C TCTCGGGC 414
13 UUGCCCGA CUGAUGAG X CGAA AGAGACGA3 TCGTCTCT C TCGGGCAA 415
15 UGUUGCCC CUGAUGAG X CGAA AGAGAGAC4 GTCTCTCT C GGGCAACA 416
43 GAGGCCGC CUGAUGAG X CGAA ACCUCCUC5 GAGGAGGT A GCGGCCTC 417
51 GGCUCCCG CUGAUGAG X CGAA AGGCCGCU6 AGCGGCCT C CGGGAGCC 418
80 GUCGUCUG CUGAUGAG X CGAA AUCCAGGU7 ACCTGGAT C CAGACGAC 419
108 CAGCCGUA CUGAUGAG X CGAA AGGCAGCU8 AGCTGCCT C TACGGCTG 420
110 CUCAGCCG CUGAUGAG X CGAA AGAGGCAG9 CTGCCTCT A CGGCTGAG 421
167 UGCUGCAG CUGAUGAG X CGAA AGGCCCUU10 AAGGGCCT T CTGCAGCA 422
168 CUGCUGCA CUGAUGAG X CGAA AAGGCCCU11 AGGGCCTT C TGCAGCAG 423
194 UGAUUCUU CUGAUGAG X CGAA AUCAGGUU12 AACCTGAT A AAGAATCA 424
201 AGGCUCCU CUGAUGAG X CGAA AUUCUUUA13 TAAAGAAT C AGGAGCCT 425
210 CAUCCACU CUGAUGAG X CGAA AGGCUCCU14 AGGAGCCT C AGTGGATG 426
223 UGUCUUGC CUGAUGAG X CGAA ACUUCAUC15 GATGAAGT A GCAAGACA 427
234 AUCUUUCC CUGAUGAG X CGAA ACUGUCUU16 AAGACAGT T GGAAAGAT 428
243 CCAAUGCU CUGAUGAG X CGAA AUCUUUCC17 GGAAAGAT C AGCATTGG 429
249 UAUCUUCC CUGAUGAG X CGAA AUGCUGAU18 ATCAGCAT T GGAAGATA 430
257 UCUUUCUU CUGAUGAG X CGAA AUCUUCCA19 TGGAAGAT A AAGAAAGA 431
355 UCUGUUUG CUGAUGAG X CGAA ACUUUUGG20 CCAAAAGT T CAAACAGA 432
356 GUCUGUUU CUGAUGAG X CGAA AACUUUUG21 CAAAAGTT C AAACAGAC 433
368 AACUGAGG CUGAUGAG X CGAA AGGGUCUG22 CAGACCCT C.CCTCAGTT 434
372 UUGGAACU CUGAUGAG X CGAA AGGGAGGG23 CCCTCCCT C AGTTCCAA 435
376 CAUAUUGG CUGAUGAG X CGAA ACUGAGGG24 CCCTCAGT T CCAATATG 436
377 ACAUAUUG CUGAUGAG X CGAA AACUGAGG25 CCTCAGTT C CAATATGT 437
382 AGGUCACA CUGAUGAG X CGAA AUUGGAAC26 GTTCCAAT A TGTGACCT 438
393 CAUUAGGA CUGAUGAG X CGAA ACAGGUCA.27 TGACCTGT A TCCTAATG 439
395 ACCAUUAG CUGAIJGAG X CGAA AUACAGGU28 ACCTGTAT C CTAATGGT 440
398 UACACCAU CUGAUGAG X CGAA AGGAUACA29 TGTATCCT A ATGGTGTA 441
406 UUGGGAAA CUGAUGAG X CGAA ACACCAUU30 AATGGTGT A TTTCCCAA 442
408 CUUUGGGA CUGAUGAG X CGAA AUACACCA31 TGGTGTAT T TCCCAAAG 443
409 CCUUUGGG CUGAUGAG X CGAA AAUACACC32 GGTGTATT T CCCAAAGG 444
410 UCCUUUGG CUGAUGAG X CGAA AAAUACAC33 GTGTATTT C CCAAAGGA 445
432 UGGGUGGG CUGAUGAG X CGAA AUUCGCAU34 ATGCGAAT A CCCACCCA 446
464 AGUUCUCC CUGAUGAG X CGAA AGCAGCUG35 CAGCTGCT T GGAGAACT 447
473 UUCACUUG CUGAUGAG X CGAA AGWCUCC36 GGAGAACT A CAAGTGAA 448
495 CCUGAUCU CUGAUGAG X CGAA AUGCUUUC37 GAAAGCAT T AGATCAGG 449
496 GCCUGAUC CUGAUGAG X CGAA AAUGCUUU38 AAAGCATT A GATCAGGC 450
500 ACUUGCCU CUGAUGAG X CGAA AUCUAAUG39 CATTAGAT C AGGCAAGT 451
517 UCAUUCCA CUGAUGAG X CGAA AUCUCUUC40 GAAGAGAT T TGGAATGA 452
518 AUCAUUCC CUGAUGAG X CGAA AAUCUCUU41 AAGAGATT T GGAATGAT 453
527 UUCUCGAA CUGAUGAG X CGAA AUCAUUCC42 GGAATGAT T TTCGAGAA ~ 4541
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Table 9
528 CUUCUCGA CUGAUGAG X CGAA AAUCAUUC43 GAATGATT T TCGAGAAG 455
529 GCUUCUCG CUGAUGAG X CGAA AAAUCAUU44 AATGATTT T CGAGAAGC 456
530 AGCUUCUC CUGAUGAG X CGAA AAAAUCAU45 ATGATTTT C GAGAAGCT 457
551 AACUUGUC CUGALJGAG X CGAA AUGUGCUU46 AAGCACAT C GACAAGTT 458
559 UAUUUUCU CUGAUGAG X CGAA ACUUGUCG47 CGACAAGT T AGAAAATA 459
560 GUAUUUUC CUGAUGAG X CGAA AACUUGUC48 GACAAGTT A GAAAATAC 460
567 UCAUUACG CUGAUGAG X CGAA AUUUUCUA49 TAGAAAAT A CGTAATGA 461
571 CAGCUCAU CUGAUGAG X CGAA ACGUAUUU50 AAATACGT A ATGAGCTG 462
583 CCAGGCUU CUGAUGAG X CGAA AUCCAGCU51 AGCTGGAT C AAGCCTGG 463
604 ~ CAGAUUUC CUGAUGAG X CGAA 52 ACAATGAT A GAAATCTG 464
AUCAUUGU
610 UUUUCACA CUGAUGAG X CGAA AUUUCUAU53 ATAGAAAT C TGTGAAAA 465
621 AGUCUUCC CUGAUGAG X CGAA ACUUUUCA54 TGAAAAGT T GGAAGACT 466
632 CUUGCGUG CUGAUGAG X CGAA ACAGUCUU55 AAGACTGT T CACGCAAG 467
633 ACUUGCGU CUGAUGAG X CGAA AACAGUCU56 AGACTGTT C ACGCAAGT 468
642 CUUUUAUU CUGAUGAG X CGAA ACUUGCGU57 ACGCAAGT T AATAAAAG 469
643 UCUUUUAU CUGAUGAG X CGAA AACUUGCG58 CGCAAGTT A ATAAAAGA 470
646 UUCUCUUU CUGAUGAG X CGAA AUUAACUU59 AAGTTAAT A AAAGAGAA 471
660 CUGCAUUU CUGAUGAG X CGAA AUCCAUUC60 GAATGGAT T AAATGCAG 472
661 CCUGCAUU CUGAUGAG X CGAA AAUCCAUU61 AATGGATT A AATGCAGG 473
678 CAGUAGGA CUGAUGAG X CGAA AUGCCAGG62 CCTGGCAT T TCCTACTG 474
679 CCAGUAGG CUGAUGAG X CGAA AAUGCCAG63 CTGGCATT T CCTACTGG 475
680 UCCAGUAG CUGAUGAG X CGAA AAAUGCCA64 TGGCATTT C CTACTGGA 476
683 ACAUCCAG CUGAUGAG X CGAA AGGAAAUG65 CATTTCCT A CTGGATGT 477
692 AUUGAGAG CUGAUGAG X CGAA ACAUCCAG66 CTGGATGT T CTCTCAAT 478
693 UAUUGAGA CUGAUGAG X CGAA AACAUCCA67 TGGATGTT C TCTCAATA 479
'
695 AUUAUUGA CUGAUGAG X CGAA AGAACAUC68 GATGTTCT C TCAATAAT 480
697 CAAUUAUU CUGAUGAG X CGAA AGAGAACA69 TGTTCTCT C AATAATTG 481
701 AGCACAAU CUGAUGAG X CGAA AUUGAGAG70 CTCTCAAT A ATTGTGCT 482
704 GGCAGCAC CUGAUGAG X CGAA AUUAUUGA71 TCAATAAT T GTGCTGCC 483
716 GGGAGUAU CUGAUGAG X CGAA AUGGGCAG72 CTGCCCAT T ATACTCCC 484
717 UGGGAGUA CUGAUGAG X CGAA AAUGGGCA73 TGCCCATT A TACTCCCA 485
719 AUUGGGAG CUGAUGAG X CGAA AUAAUGGG74 CCCATTAT A CTCCCAAT 486
722 GGCAUUGG CUGAUGAG X CGAA AGUAUAAU75 ATTATACT C CCAATGCC 487
745 UACUGUAA CUGAUGAG X CGAA ACUGUUGU76 ACAACAGT A TTACAGTA 488
747 CAUACUGU CUGAUGAG X CGAA AUACUGUU77 AACAGTAT T ACAGTATG 489
748 UCAUACUG CUGAUGAG X CGAA AAUACUGU7B ACAGTATT A CAGTATGA 490
753 UGUCAUCA CUGAUGAG X CGAA ACUGUAAU79 ATTACAGT A TGATGACA 491
763 AUUUUACA CUGAUGAG X CGAA AUGUCAUC80 GATGACAT C TGTAAAAT 492
767 GUCUAUUU CUGAUGAG X CGAA ACAGAUGU81 ACATCTGT A AAATAGAC 493
772 CCAAAGUC CUGAUGAG X CGAA AUUUUACA82 TGTAAAAT A GACTTTGG 494
777 GUGUUCCA CUGAUGAG X CGAA AGUCUAUU83 AATAGACT T TGGAACAC 495
778 UGUGUUCC CUGAUGAG X CGAA AAGUCUAU84 ATAGACTT T GGAACACA 496
788 ACCACUUA CUGAUGAG X CGAA AUGUGUUC85 GAACACAT A TAAGTGGT 497
790 CUACCACU CUGAUGAG X CGAA AUAUGUGU86 ACACATAT A AGTGGTAG 498
797 AAUAAUCC CUGAUGAG X CGAA ACCACUUA87 TAAGTGGT A GGATTATT 499
802 CAGUCAAU CUGAUGAG X CGAA AUCCUACC88 GGTAGGAT T ATTGACTG 500
803 ACAGUCAA CUGAUGAG X CGAA AAUCCUAC89 GTAGGATT A TTGACTGT 501
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Table 9
805 GCACAGUC CUGAUGAG X CGAA AUAAUCCU90 AGGATTAT T GACTGTGC 502
815 GACAGUAA CUGAUGAG X CGAA AGCACAGU91 ACTGTGCT T TTACTGTC 503
816 UGACAGUA CUGAUGAG X CGAA AAGCACAG92 CTGTGCTT T TACTGTCA 504
817 GUGACAGU CUGAUGAG X CGAA AAAGCACA93 TGTGCTTT T ACTGTCAC 505
818 AGUGACAG CUGAUGAG X CGAA AAAAGCAC94 GTGCTTTT A CTGTCACT 506
823 UUAAAAGU CUGAUGAG X CGAA ACAGUAAA95 TTTACTGT C ACTTTTAA 507
827 GGGAUUAA CUGAUGAG X CGAA AGUGACAG96 CTGTCACT T TTAATCCC 508
828 UGGGAUUA CUGAUGAG X CGAA AAGUGACA97 TGTCACTT T TAATCCCA 509
829 UUGGGAUU CUGAUGAG X CGAA AAAGUGAC98 GTCACTTT T AATCCCAA 510
830 UUUGGGAU CUGAUGAG X CGAA AAAAGUGA99 TCACTTTT A ATCCCAAA 511
833 AUAUUUGG CUGAUGAG X CGAA AUUAAAAG100 CTTTTAAT C CCAAATAT 512
840 ACGUAUCA CUGAUGAG X CGAA AUUUGGGA101 TCCCAAAT A TGATACGT 513
845 UAAUAACG CUGAUGAG X CGAA AUCAUAUU102 AATATGAT A CGTTATTA 514
849 CUUUUAAU CUGAUGAG X CGAA ACGUAUCA103 TGATACGT T ATTAAAAG 515
850 GCUUUUAA CUGAUGAG X CGAA AACGUAUC104 GATACGTT A TTAAAAGC 516
852 CAGCUUW CUGAUGAG X CGAA AUAACGUA105 TACGTTAT T AAAAGCTG 517
853 ACAGCUUU CUGAUGAG X CGAA AAUAACGU106 ACGTTATT A AAAGCTGT 518
862 GCAUCUUU CUGAUGAG X CGAA ACAGCUW107 AAAGCTGT A AAAGATGC 519
872 AGUGUUAG CUGAUGAG X CGAA AGCAUCUU108 AAGATGCT A CTAACACT 520
875 UCCAGUGU CUGAUGAG X CGAA AGUAGCAU109 ATGCTACT A ACACTGGA 521
886 GCACACUU CUGAUGAG X CGAA AUUCCAGU110 ACTGGAAT A AAGTGTGC 522
901 CGAACAUC CUGAUGAG X CGAA AUUCCAGC111 GCTGGAAT T GATGTTCG 523
907 CACAGACG CUGAUGAG X CGAA ACAUCAAU112 ATTGATGT T CGTCTGTG 524
908 ACACAGAC CUGAUGAG X CGAA AACAUCAA113 TTGATGTT C GTCTGTGT 525
911 AUCACACA CUGAUGAG X CGAA ACGAACAU114 ATGTTCGT C TGTGTGAT 526
922 GCCUCACC CUGAUGAG X CGAA ACAUCACA115 TGTGATGT T GGTGAGGC 527
934 ACUUCUUG CUGAUGAG X CGAA AUGGCCUC116 GAGGCCAT C CAAGAAGT 528
943 GACUCCAU CUGAUGAG X CGAA ACUUCUUG117 CAAGAAGT T ATGGAGTC 529
944 GGACUCCA CUGAUGAG X CGAA AACUUCUU118 AAGAAGTT A TGGAGTCC 530
951 CUUCAUAG CUGAUGAG X CGAA ACUCCAUA119 TATGGAGT C CTATGAAG 531
954 CAACUUCA CUGAUGAG X CGAA AGGACUCC120 GGAGTCCT A TGAAGTTG 532
961 UCUAUUUC CUGAUGAG X CGAA ACUUCAUA121 TATGAAGT T GAAATAGA 533
967 UUCCCAUC CUGAUGAG X CGAA AUUUCAAC122 GTTGAAAT A GATGGGAA 534
981 UCACUUGA CUGALJGAG X CGAA AUGUCUUC123 GAAGACAT A TCAAGTGA 535
983 UUUCACW CUGAUGAG X CGAA AUAUGUCU124 AGACATAT C AAGTGAAA 536
997 AGAUUACG CUGAUGAG X CGAA AUUGGUUU125 AAACCAAT C CGTAATCT 537
1001 AUUUAGAU CUGAUGAG X CGAA ACGGAUUG126 CAATCCGT A ATCTAAAT 538
1004 UCCAUUUA CUGAUGAG X CGAA AUUACGGA127 TCCGTAAT C TAAATGGA 539
1006 UGUCCAUU CUGAUGAG X CGAA AGAUUACG128 CGTAATCT A AATGGACA 540
1016 CCCAAUUG CUGAUGAG X CGAA AUGUCCAU129 ATGGACAT T CAATTGGG 541
1017 GCCCAAUU CUGAUGAG X CGAA AAUGUCCA130 TGGACATT C AATTGGGC 542
1021 UAUUGCCC CUGAUGAG X CGAA AUUGAAUG131 CATTCAAT T GGGCAATA 543
1029 GUAUUCUA CUGAUGAG X CGAA AWGCCCA132 TGGGCAAT A TAGAATAC 544
1031 AUGUAUUC CUGAUGAG X CGAA AUAUUGCC.133 GGCAATAT A GAATACAT 545
1036 CCAGCAUG CUGAUGAG X CGAA AUUCUAUA134 TATAGAAT A CATGCTGG 546
-
1060 CCUUUCAC CUGAUGAG X CGAA AUCGGCAC135 GTGCCGAT T GTGAAAGG 547
1102 AUUGCAUA CUGAUGAG X CGAA ACUUCUCC136 GGAGAAGT A TATGCAAT I 5481
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1104 CAAUUGCA CUGAUGAG X CGAA AUACUUCU137 AGAAGTAT A TGCAATTG 549
1111 AAGGUUUC CUGAUGAG X CGAA AUUGCAUA138 TATGCAAT T GAAACCTT 550
1119 UACUACCA CUGAUGAG X CGAA AGGUUUCA139 TGAAACCT T TGGTAGTA 551
1120 GUACUACC CUGAUGAG X CGAA AAGGUUUC140 GAAACCTT T GGTAGTAC 552
1124 UCCUGUAC CUGAUGAG X CGAA ACCAAAGG141 CCTTTGGT A GTACAGGA 553
1127 UUUUCCUG CUGAUGAG X CGAA ACUACCAA142 TTGGTAGT A CAGGAAAA 554
1141 UCAUGAAC CUGAUGAG X CGAA ACACCUUU143 AAAGGTGT T GTTCATGA 555
1144 UCAUCAUG CUGAUGAG X CGAA ACAACACC144 GGTGTTGT T CATGATGA 556
1145 AUCAUCAU CUGAUGAG X CGAA AACAACAC145 GTGTTGTT C ATGATGAT 557
1154 ACAUUCCA CUGAUGAG X CGAA AUCAUCAU146 ATGATGAT A TGGAATGT 558
1163 GUAAUGUG CUGAUGAG X CGAA ACAUUCCA147 TGGAATGT T CACATTAC 559
1164 UGUAAUGU CUGAUGAG X CGAA AACAUUCC148 GGAATGTT C ACATTACA 560
1169 UUUCAUGU CUGAUGAG X CGAA AUGLTGAAC149 GTTCACAT T ACATGAAA 561
1170 UUUUCAUG CUGAUGAG X CGAA AAUGUGAA150 TTCACATT A CATGAAAA 562
1181 AACAUCAA CUGAUGAG X CGAA AUUUUUCA151 TGAAAAAT T TTGATGTT 563
1182 CAACAUCA CUGAUGAG X CGAA AAUUUUUC152 GAAAAATT T TGATGTTG 564
1183 CCAACAUC CUGAUGAG X CGAA AAAUUUUU153 AAAAATTT T GATGTTGG 565
1189 ACAUGUCC CUGAUGAG X CGAA ACAUCAAA154 TTTGATGT T GGACATGT 566
1204 GGAAGCCU CUGAUGAG X CGAA AUUGGCAC155 GTGCCAAT A AGGCTTCC 567
1210 GUUCUUGG CUGAUGAG X CGAA AGCCUUAU156 ATAAGGCT T CCAAGAAC 568
1211 UGUUCUUG CUGAUGAG X CGAA AAGCCUUA157 TAAGGCTT C CAAGAACA 569
1227 CAUUUAAC CUGAUGAG X CGAA AGUGUUUU158 AAAACACT T GTTAAATG 570
1230 UGACAUUU CUGAUGAG X CGAA ACAAGUGU159 ACACTTGT T AAATGTCA 571
1231 AUGACAUU CUGAUGAG X CGAA AACAAGUG160 CACTTGTT A AATGTCAT 572
1237 UCAUUGAU CUGAUGAG X CGAA ACAUUUAA161 TTAAATGT C ATCAATGA 573
1240 UUUUCAUU CUGAUGAG X CGAA AUGACAUU162 AATGTCAT C AATGAAAA 574
1251 GGGUUCCA CUGAUGAG X CGAA AGUUUUCA163 TGAAAACT T TGGAACCC 575
1252 AGGGUUCC CUGAUGAG X CGAA AAGUUUUC164 GAAAACTT T GGAACCCT 576
1261 CAGAAGGC CUGAUGAG X CGAA AGGGUUCC165 GGAACCCT T GCCTTCTG 577
1266 UGCGGCAG CUGAUGAG X CGAA AGGCAAGG166 CCTTGCCT T CTGCCGCA 578
1267 CUGCGGCA CUGAUGAG X CGAA AAGGCAAG167 CTTGCCTT C TGCCGCAG 579
1286 UCCCAAGC CUGAUGAG X CGAA AUCCAGCC168 GGCTGGAT C GCTTGGGA 580
1290 UUUCUCCC CUGAUGAG X CGAA AGCGAUCC169 GGATCGCT T GGGAGAAA 581
1301 CAAGUAUU CUGAUGAG X CGAA ACUUUCUC170 GAGAAAGT A AATACTTG 582
1305 CCAUCAAG CUGAUGAG X CGAA AUUUACUU171 AAGTAAAT A CTTGATGG 583
1308 GAGCCAUC CUGAUGAG X CGAA AGUAUUUA172 TAAATACT T GATGGCTC 584
1316 AUUCWCA CUGAUGAG X CGAA AGCCAUCA173 TGATGGCT C TGAAGAAT 585
1325 GUCACACA CUGAUGAG X CGAA AUUCUUCA174 TGAAGAAT C TGTGTGAC 586
1335 CAAUGCCC CUGAUGAG X CGAA AGUCACAC175 GTGTGACT T GGGCATTG 587
1342 GGAUCUAC CUGAUGAG X CGAA AUGCCCAA176 TTGGGCAT T GTAGATCC 588
1345 UAUGGAUC CUGAUGAG X CGAA ACAAUGCC177 GGCATTGT A GATCCATA 589
1349 UGGAUAUG CUGAUGAG X CGAA AUCUACAA178 TTGTAGAT C CATATCCA 590
1353 AUGGUGGA CUGAUGAG X CGAA AUGGAUCU179 AGATCCAT A TCCACCAT 591
1355 UAAUGGUG CUGAUGAG X CGAA AUAUGGAU180 ATCCATAT C CACCATTA 592
1362 UGUCACAU CUGAUGAG X CGAA AUGGUGGA181 TCCACCAT T ATGTGACA 593'
1363 AUGUCACA CUGAUGAG X CGAA AAUGGUGG182 CCACCATT A TGTGACAT 594
1372 GAUCCUUU CUGAUGAG X CGAA AUGUCACA183 TGTGACAT T AAAGGATC 595
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1373 UGAUCCUU CUGAUGAG X CGAA AAUGUCAC184 GTGACATT A AAGGATCA 596
1380 CUGUAUAU CUGAUGAG X CGAA AUCCUUUA185 TAAAGGAT C ATATACAG 597
1383 GCGCUGUA CUGAUGAG X CGAA AUGAUCCU186 AGGATCAT A TACAGCGC 598
1385 UUGCGCUG CUGAUGAG X CGAA AUAUGAUC187 GATCATAT A CAGCGCAA 599
1395 UAUGUUCA CUGAUGAG X CGAA AUUGCGCU188 AGCGCAAT T TGAACATA 600
1396 GUAUGUUC CUGAUGAG X CGAA AAUUGCGC189 GCGCAATT T GAACATAC 601
1403 CAGGAUGG CUGAUGAG X CGAA AUGUUCAA190 TTGAACAT A CCATCCTG 602
1408 CGCAACAG CUGAUGAG X CGAA AUGGUAUG191 CATACCAT C CTGTTGCG 603
1413 UUGGACGC CUGAUGAG X CGAA ACAGGAUG192 CATCCTGT T GCGTCCAA 604
1418 ACAUGUUG CUGAUGAG X CGAA ACGCAACA193 TGTTGCGT C CAACATGT 605
1427 AACUUCUU CUGAUGAG X CGAA ACAUGUUG194 CAACATGT A AAGAAGTT 606
1435 CUGCUGAC CUGAUGAG X CGAA ACUUCUUU195 AAAGAAGT T GTCAGCAG 607
1438 CCUCUGCU CUGAUGAG X CGAA ACAACUUC196 GAAGTTGT C AGCAGAGG 608
1455 AAGUUUAA CUGAUGAG X CGAA AGUCAUCU197 AGATGACT A TTAAACTT 609
1457 CUAAGUUU CUGAUGAG X CGAA AUAGUCAU198 ATGACTAT T AAACTTAG 610
1458 ACUAAGUU CUGAUGAG X CGAA AAUAGUCA199 TGACTATT A AACTTAGT 611
1463 UUUGGACU CUGAUGAG X CGAA AGUUUAAU200 ATTAAACT T AGTCCAAA 612
1464 CUUUGGAC CUGAUGAG X CGAA AAGUUUAA201 TTAAACTT A GTCCAAAG 613
1467 UGGCUUUG CUGAUGAG X CGAA ACUAAGUU202 AACTTAGT C CAAAGCCA 614
1479 AAGGUGUU CUGAUGAG X CGAA AGGUGGCU203 AGCCACCT C AACACCTT 615
1487 AGAAAAUA CUGAUGAG X CGAA AGGUGUUG204 CAACACCT T TATTTTCT 616
1488 CAGAAAAU CUGAUGAG X CGAA AAGGUGUU205 AACACCTT T ATTTTCTG 617
1489 UCAGAAAA CUGAUGAG X CGAA AAAGGUGU206 ACACCTTT A TTTTCTGA 618
1491 GCUCAGAA CUGAUGAG X CGAA AUAAAGGU207 ACCTTTAT T TTCTGAGC 619
1492 AGCUCAGA CUGAUGAG X CGAA AAUAAAGG208 CCTTTATT T TCTGAGCT 620
'
1493 AAGCUCAG CUGAUGAG X CGAA AAAUAAAG2 CTTTATTT T CTGAGCTT 621
09
1494 AAAGCUCA CUGAUGAG X CGAA AAAAUAAA210 TTTATTTT C TGAGCTTT 622
1501 UUCCAACA CUGAUGAG X CGAA AGCUCAGA211 TCTGAGCT T TGTTGGAA 623
1502 UWCCAAC CUGAUGAG X CGAA AAGCUCAG212 CTGAGCTT T GTTGGAAA 624
1505 UGUUWCC CUGAUGAG X CGAA ACAAAGCU213 AGCTTTGT T GGAAAACA 625
1518 AAUUCUGG CUGAUGAG X CGAA AUCAUGUU214 AACATGAT A CCAGAATT 626
1526 GGCAAAUU CUGAUGAG X CGAA AUUCUGGU215 ACCAGAAT T AATTTGCC 627
1527 UGGCAAAU CUGAUGAG X CGAA AAUUCUGG216 CCAGAATT A ATTTGCCA 628
1530 AUGUGGCA CUGAUGAG X CGAA AWAAUUC217 GAATTAAT T TGCCACAT 629
1531 CAUGUGGC CUGAUGAG X CGAA AAUUAAUU218 AATTAATT T GCCACATG 630
1541 AAACAGAC CUGAUGAG X CGAA ACAUGUGG219 CCACATGT T GTCTGTTT 631
1544 UUAAAACA CUGAUGAG X CGAA ACAACAUG220 CATGTTGT C TGTTTTAA 632
1548 ACUGUUAA CUGAUGAG X CGAA ACAGACAA221 TTGTCTGT T TTAACAGT 633
1549 CACUGUUA CUGAUGAG X CGAA AACAGACA222 TGTCTGTT T TAACAGTG 634
1550 CCACUGUU CUGAUGAG X CGAA AAACAGAC223 GTCTGTTT T AACAGTGG 635
1551 UCCACUGU CUGAUGAG X CGAA AAAACAGA224 TCTGTTTT A ACAGTGGA 636
1567 AAAAGUAU CUGAUGAG X CGAA ACAUGGGU225 ACCCATGT A ATACTTTT 637
1570 GAUAAAAG CUGAUGAG X CGAA AUUACAUG226 CATGTAAT A CTTTTATC 638
1573 AUGGAUAA CUGAUGAG X CGAA AGUAUUAC227 GTAATACT T TTATCCAT 639
1574 CAUGGAUA CUGAUGAG X CGAA AAGUAUUA228 TAATACTT T TATCCATG 640
1575 ACAUGGAU CUGAUGAG X CGAA AAAGUAUU229 AATACTTT T ATCCATGT 641
1576 AACAUGGA CUGAUGAG X CGAA AAAAGUAU230 ATACTTTT A TCCATGTT 6421
I
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1578 UAAACAUG CUGAUGAG X CGAA AUAAAAGU231 ACTTTTAT C CATGTTTA 643
1584 CUUWUUA CUGAUGAG X CGAA ACAUGGAU232 ATCCATGT T TAAAAAAG 644
1585 UCUUUUfIU CUGAUGAG X CGAA AACAUGGA233 TCCATGTT T AAAAAAGA 645
1586 UUCUUUUU CUGAUGAG X CGAA AAACAUGG234 CCATGTTT A AAAAAGAA 646
1600 UWGUCCA CUGAUGAG X CGAA AUUCCUUC235 GAAGGAAT T TGGACAAA 647
1601 CUUUGUCC CUGAUGAG X CGAA AAUUCCUU236 AAGGAATT T GGACAAAG 648
1619 UUACAUUA CUGAUGAG X CGAA ACGGUUUG237 CAAACCGT C TAATGTAA 649
1621 AAWACAU CUGAUGAG X CGAA AGACGGUU238 AACCGTCT A ATGTAATT 650
1626 UGGWAAU CUGAUGAG X CGAA ACAUUAGA239 TCTAATGT A ATTAACCA 651
1629 CGUUGGUU CUGAUGAG X CGAA AUUACAUU240 AATGTAAT T AACCAACG 652
1630 UCGUUGGU CUGAUGAG X CGAA AAUUACAU241 ATGTAATT A ACCAACGA 653
1646 AGUCCGGA CUGAUGAG X CGAA AGCUUUUU242 AAAAAGCT T TCCGGACT 654
1647 AAGUCCGG CUGAUGAG X CGAA AAGCUUUU243 AAAAGCTT T CCGGACTT 655
,1648 AAAGUCCG CUGAUGAG X CGAA AAAGCUUU244 AAAGCTTT C CGGACTTT 656
1655 GCAUUUAA CUGAUGAG X CGAA AGUCCGGA245 TCCGGACT T TTAAATGC 657
1656 AGCAUUUA CUGAUGAG X CGAA AAGUCCGG246 CCGGACTT T TAAATGCT 658
1657 UAGCAUUU CUGAUGAG X CGAA AAAGUCCG247 CGGACTTT T AAATGCTA 659
1658 UUAGCAUU CUGAUGAG X CGAA AAAAGUCC248 GGACTTTT A AATGCTAA 660
1665 AAAACAGU CUGAUGAG X CGAA AGCAUUUA249 TAAATGCT A ACTGTTTT 661
1671 AGGGGAAA CUGAUGAG X CGAA ACAGUUAG250 CTAACTGT T TTTCCCCT 662
1672 AAGGGGAA CUGAUGAG X CGAA AACAGUUA251 TAACTGTT T TTCCCCTT 663
1673 GAAGGGGA CUGAUGAG X CGAA AAACAGUU252 AACTGTTT T TCCCCTTC 664
1674 GGAAGGGG CUGAUGAG X CGAA AAAACAGU253 ACTGTTTT T CCCCTTCC 665
1675 AGGAAGGG CUGAUGAG X CGAA AAAAACAG254 CTGTTTTT C CCCTTCCT 666
1680 UAGACAGG CUGAUGAG X CGAA AGGGGAAA255 TTTCCCCT T CCTGTCTA 667
1681 CUAGACAG CUGAUGAG X CGAA AAGGGGAA256 TTCCCCTT C CTGTCTAG 668
1686 UWUCCUA CUGAUGAG X CGAA ACAGGAAG257 CTTCCTGT C TAGGAAAA 669
1688 CAUUUUCC CUGAUGAG X CGAA AGACAGGA258 TCCTGTCT A GGAAAATG 670
1699 GAGCUUUA CUGAUGAG X CGAA AGCAUUUU259 AAAATGCT A TAAAGCTC 671
1701 UUGAGCUU CUGAUGAG X CGAA AUAGCAUU260 AATGCTAT A AAGCTCAA 672
1707 ACUAAUUU CUGAUGAG X CGAA AGCUUUAU261 ATAAAGCT C AAATTAGT 673
1712 UCCUAACU CUGAUGAG X CGAA AUWGAGC262 GCTCAAAT T AGTTAGGA 674
1713 UUCCUAAC CUGAUGAG X CGAA AAUUUGAG263 CTCAAATT A GTTAGGAA 675
1716 UCAUUCCU CUGAUGAG X CGAA ACUAAUUU264 AAATTAGT T AGGAATGA 676
1717, GUCAUUCC CUGAUGAG X CGAA AACUAAUU265 AATTAGTT A GGAATGAC 677
1727 AAACGUAU CUGAUGAG X CGAA AGUCAUUC266 GAATGACT T ATACGTTT 678
1728 AAAACGUA CUGAUGAG X CGAA AAGUCAUU267 AATGACTT A TACGTTTT 679
1730 ACAAAACG CUGAUGAG X CGAA AUAAGUCA268 TGACTTAT A CGTTTTGT 680
1734 CAAAACAA CUGAUGAG X CGAA ACGUAUAA269 TTATACGT T TTGTTTTG 681
1735 UCAAAACA CUGAUGAG X CGAA AACGUAUA270 TATACGTT T TGTTTTGA 682
1736 UUCAAAAC CUGAUGAG X CGAA AAACGUAU271 ATACGTTT T GTTTTGAA 683
1739 GUAUUCAA CUGAUGAG X CGAA ACAAAACG272 CGTTTTGT T TTGAATAC 684
1740 GGUAUUCA CUGAUGAG X CGAA AACAAAAC273 GTTTTGTT T TGAATACC 685
1741 AGGUAUUC CUGAUGAG X CGAA AAACAAAA274 TTTTGTTT T GAATACCT 686
1746 CUCUUAGG CUGAUGAG X CGAA AUUCAAAA275 TTTTGAAT A CCTAAGAG 687
1750 GUAUCUCU CUGAUGAG X CGAA AGGUAUUC276 GAATACCT A AGAGATAC 688
1757 CCAAAAAG CUGAUGAG X CGAA AUCUCUUA277 TAAGAGAT A CTTTTTGG 689
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i 1760 UAUCCAAA CUGAUGAG X CGAA AGUAUCUC278 GAGATACT T TTTGGATA 690
1761 AUAUCCAA CUGAUGAG X CGAA AAGUAUCU279 AGATACTT T TTGGATAT 691
1762 AAUAUCCA CUGAUGAG X CGAA AAAGUAUC280 GATACTTT T TGGATATT 692
1763 AAAUAUCC CUGAUGAG X CGAA AAAAGUAU281 ATACTTTT T GGATATTT 693
1768 AAUAUAAA CUGAUGAG X CGAA AUCCAAAA282 TTTTGGAT A TTTATATT 694
1770 GCAAUAUA CUGAUGAG X CGAA AUAUCCAA283 TTGGATAT T TATATTGC 695
1771 GGCAAUAU CUGAUGAG X CGAA AAUAUCCA284 TGGATATT T ATATTGCC 696
1772 UGGCAAUA CUGAUGAG X CGAA AAAUAUCC285 GGATATTT A TATTGCCA 697
1774 UAUGGCAA CUGAUGAG X CGAA AUAAAUAU286 ATATTTAT A TTGCCATA 698
1776 AAUAUGGC CUGAUGAG X CGAA AUAUAAAU287 ATTTATAT T GCCATATT 699
1782 AGUAAGAA CUGAUGAG X CGAA AUGGCAAU288 ATTGCCAT A TTCTTACT 700
1784 CAAGUAAG CUGAUGAG X CGAA AUAUGGCA289 TGCCATAT T CTTACTTG 701
1785 UCAAGUAA CUGAUGAG X CGAA AAUAUGGC290 GCCATATT C TTACTTGA 702
1787 AUUCAAGU CUGAUGAG X CGAA AGAAUAUG291 CATATTCT T ACTTGAAT 703
1788 CAUUCAAG CUGAUGAG X CGAA AAGAAUAU292 ATATTCTT A CTTGAATG 704
1791 AAGCAUUC CUGAUGAG X CGAA AGUAAGAA293 TTCTTACT T GAATGCTT 705
1799 GUCAUUCA CUGAUGAG X CGAA AGCAUUCA294 TGAATGCT T TGAATGAC 706
1800 AGUCAUUC CUGAUGAG X CGAA AAGCAUUC295 GAATGCTT T GAATGACT 707
1809 ACUGGAUG CUGAUGAG X CGAA AGUCAUUC296 GAATGACT A CATCCAGT 708
1813 CAGAACUG CUGAUGAG X CGAA AUGUAGUC297 GACTACAT C CAGTTCTG 709
1818 AGGUGCAG CUGAUGAG X CGAA ACUGGAUG298 CATCCAGT T CTGCACCT 710
1819 UAGGUGCA CUGAUGAG X CGAA AACUGGAU299 ATCCAGTT C TGCACCTA 711
1827 AGAGGGUA CUGAUGAG X CGAA AGGUGCAG300 CTGCACCT A TACCCTCT 712
1829 CCAGAGGG CUGAUGAG X CGAA AUAGGUGC301 GCACCTAT A CCCTCTGG 713
1834 CAACACCA CUGAUGAG X CGAA AGGGUAUA302 TATACCCT C TGGTGTTG 714
1841 UAAAAAGC CUGAUGAG X CGAA ACACCAGA303 TCTGGTGT T GCTTTTTA 715
1845 AGGUUAAA CUGAUGAG X CGAA AGCAACAC304 GTGTTGCT T TTTAACCT 716
1846 AAGGUUAA CUGAUGAG X CGAA AAGCAACA305 TGTTGCTT T TTAACCTT 717
1847 GAAGGUUA CUGAUGAG X CGAA AAAGCAAC306 GTTGCTTT T TAACCTTC 718
1848 GGAAGGUU CUGAUGAG X CGAA AAAAGCAA307 TTGCTTTT T AACCTTCC 719
1849 AGGAAGGU CUGAUGAG X CGAA AAAAAGCA308 TGCTTTTT A ACCTTCCT 720
1854 AWCCAGG CUGAUGAG X CGAA AGGUUAAA309 TTTAACCT T CCTGGAAT 721
1855 GAUUCCAG CUGAUGAG X CGAA AAGGUUAA310 TTAACCTT C CTGGAATC 722
1863 AGAAAAUG CUGAUGAG X CGAA AUUCCAGG311 CCTGGAAT C CATTTTCT 723
1867 UUUUAGAA CUGAUGAG X CGAA AUGGAUUC312 GAATCCAT T TTCTAAAA 724
1868 UUUUUAGA CUGAUGAG X CGAA AAUGGAUU313 AATCCATT T TCTAAAAA 725
1869 UUUUUUAG CUGAUGAG X CGAA AAAUGGAU314 ATCCATTT T CTAAAAAA 726
1870 AUUUUUUA CUGAUGAG X CGAA AAAAUGGA315 TCCATTTT C TAAAAAAT 727
1872 UUAUUUW CUGAUGAG X CGAA AGAAAAUG316 CATTTTCT A AAAAATAA 728
1879 UGUGUCUU CUGAUGAG X CGAA AUUWWA317 TAAAAAAT A AAGACACA 729
1889 CUGAGAAG CUGAUGAG X CGAA AUGUGUCU318 AGACACAT T CTTCTCAG 730
1890 GCUGAGAA CUGAUGAG X CGAA AAUGUGUC319 GACACATT C TTCTCAGC 731
1892 GUGCUGAG CUGAUGAG X CGAA AGAAUGUG320 CACATTCT T CTCAGCAC 732
1893 GGUGCUGA CUGAUGAG X CGAA AAGAAUGU321 ACATTCTT C TCAGCACC 733
1895 GUGGUGCU CUGAUGAG X CGAA AGAAGAAU322 ATTCTTCT C AGCACCAC 734
1913 UUUUGGAA CUGAUGAG X CGAA AGGUGUUG323 CAACACCT A TTCCAAAA 735
1915 GAUUUUGG CUGAUGAG X CGAA AUAGGUGU324 ACACCTAT T CCAAAATC 736
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1916 CGAUUUUG CUGAUGAG X CGAA AAUAGGUG325 CACCTATT C CAAAATCG 737
1923 AUGUGGUC CUGAUGAG X CGAA AUUUUGGA326 TCCAAAAT C GACCACAT 738
1932 CUUCCAAA CUGAUGAG X CGAA AUGUGGUC327 GACCACAT A TTTGGAAG 739
1934 UACUUCCA CUGAUGAG X CGAA AUAUGUGG328 CCACATAT T TGGAAGTA 740
1935 UUACUUCC CUGAUGAG X CGAA AAUAUGUG329 CACATATT T GGAAGTAA 741
1942 GAGAGCUU CUGAUGAG X CGAA ACUUCCAA330 TTGGAAGT A AAGCTCTC 742
1948 GCUGAGGA CUGAUGAG X CGAA AGCUUUAC331 GTAAAGCT C TCCTCAGC 743
1950 UUGCUGAG CUGAUGAG X CGAA AGAGCUUU332 AAAGCTCT C CTCAGCAA 744
1953 CAUWGCU CUGAUGAG X CGAA AGGAGAGC333 GCTCTCCT C AGCAAATG 745
1963 UGUUCUUU CUGAUGAG X CGAA ACAUUUGC334 GCAAATGT A AAAGAACA 746
1977 UUUGUUAU CUGAUGAG X CGAA AUUUCUGU335 ACAGAAAT T ATAACAAA 747
1978 GUUUGUUA CUGAUGAG X CGAA AAUWCLTG336 CAGAAATT A TAACAAAC 748
1980 CAGUUUGU CUGAUGAG X CGAA AUAAUUUC337 GAAATTAT A ACAAACTG 749
1990 GUCUGAGA CUGAUGAG X CGAA ACAGUUUG338 CAAACTGT C TCTCAGAC 750
1992 UGGUCUGA CUGAUGAG X CGAA AGACAGUU339 AACTGTCT C TCAGACCA 751
1994 UGUGGUCU CUGAUGAG X CGAA AGAGACAG340 CTGTCTCT C AGACCACA 752
2005 UUUGGUUA CUGAUGAG X CGAA ACUGUGGU341 ACCACAGT A TAACCAAA 753
2007 AGUUUGGU CUGAUGAG X CGAA AUACUGUG342 CACAGTAT A ACCAAACT 754
2016 CUGAGUUC CUGAUGAG X CGAA AGUUUGGU343 ACCAAACT A GAACTCAG 755
2022 UUAAUCCU CUGAUGAG X CGAA AGUUCUAG344 CTAGAACT C AGGATTAA 756
2028 AGUUUCUU CUGAUGAG X CGAA AUCCUGAG345 CTCAGGAT T AAGAAACT 757
2029 GAGUUUCU CUGAUGAG X CGAA AAUCCUGA346 TCAGGATT A AGAAACTC 758
2037 UUUUGAGU CUGAUGAG X CGAA AGUUUCUU347 AAGAAACT C ACTCAAAA 759
2041 GUGGUUUU CUGAUGAG X CGAA AGUGAGUU348 AACTCACT C AAAACCAC 760
2056 UUUCCAUG CUGAUGAG X CGAA AGUUGUGU349 ACACAACT A CATGGAAA 761
2079 UCAUUCAG CUGAUGAG X CGAA AGCAGGUU350 AACCTGCT C CTGAATGA 762
2090 GUAUCCAG CUGAUGAG X CGAA AGUCAUUC351 GAATGACT A CTGGATAC 763
2097 UUGUUAUG CUGAUGAG X CGAA AUCCAGUA352 TACTGGAT A CATAACAA 764
2101 CAUUUUGU CUGAUGAG X CGAA AUGUAUCC353 GGATACAT A ACAAAATG 765
2121 AACAUCUU CUGAUGAG X CGAA AUUUCUGC354 GCAGAAAT A AAGATGTT 766
2129 UUUUAAAG CUGAUGAG X CGAA ACAUCUUU355 AAAGATGT T CTTTAAAA 767
2130 GUUUUAAA CUGAUGAG X CGAA AACAUCUU356 AAGATGTT C TTTAAAAC 768
2132 UGGUUUUA CUGAUGAG X CGAA AGAACAUC357 GATGTTCT T TAAAACCA 769
2133 UUGGUUUU CUGAUGAG X CGAA AAGAACAU358 ATGTTCTT T AAAACCAA 770
2134 AUUGGUUU CUGAUGAG X CGAA AAAGAACA359 TGTTCTTT A AAACCAAT 771
2162 GAUUCUGG CUGAUGAG X CGAA AUGUUGUG360 CACAACAT A CCAGAATC 772
2170 GUCCCAGA CUGAUGAG X CGAA AUUCUGGU361 ACCAGAAT C TCTGGGAC 773
2172 GUGUCCCA CUGAUGAG X CGAA AGAUUCUG362 CAGAATCT C TGGGACAC 774
2183 CUGCUUUG CUGAUGAG X CGAA AUGUGUCC363 GGACACAT T CAAAGCAG 775
2184 ACUGCUUU CUGAUGAG X CGAA AAUGUGUC364 GACACATT C AAAGCAGT 776
2197 UUUCCCUC CUGAUGAG X CGAA ACACACUG365 CAGTGTGT A GAGGGAAA 777
2207 GUGCUAUA CUGAUGAG X CGAA AUUUCCCU366 AGGGAAAT T TATAGCAC 778
2208 AGUGCUAU CUGAUGAG X CGAA AAUUUCCC367 GGGAAATT T ATAGCACT 779
2209 UAGUGCUA CUGAUGAG X CGAA AAAUUUCC368 GGAAATTT A TAGCACTA 780
2211 UUUAGUGC CUGAUGAG X CGAA AUAAAUUU369 AAATTTAT A GCACTAAA 781
2217 UGGGCAUU CUGAUGAG X CGAA AGUGCUAU370 ATAGCACT A AATGCCCA 782
2244 AUUUUAGA CUGAUGAG X CGAA AUUUCCUG371 CAGGAAAT A TCTAAAAT I 7831
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Table 9
2246 CAAUUUUA CUGAUGAG X CGAA AUAUUUCC372 GGAAATAT C TAAAATTG 784
2248 GUCAAUUU CUGAUGAG X CGAA AGAUAUUU373 AAATATCT A AAATTGAC 785
2253 AGGGUGUC CUGAUGAG X CGAA AUUUUAGA374 TCTAAAAT T GACACCCT 786
2262 UGUGAUGU CUGAUGAG X CGAA AGGGUGUC375 GACACCCT A ACATCACA 787
2267 UUAAUUGU CUGAUGAG X CGAA AUGUUAGG376 CCTAACAT C ACAATTAA 788
2273 GUUCUUUU CUGAUGAG X CGAA AUUGUGAU377 ATCACAAT T AAAAGAAC 789
2274 AGUUCUUU CUGAUGAG X CGAA AAUUGUGA378 TCACAATT A AAAGAACT 790
2283 UGCUUCUC CUGAUGAG X CGAA AGUUCUUU379 AAAGAACT A GAGAAGCA 791
2305 AGCUUUUC CUGAUGAG X CGAA AUGUGUUU380 AAACACAT T GAAAAGCT 792
2314 CCUUCUCU CUGAUGAG X CGAA AGCUUUUC381 GAAAAGCT A AGAGAAGG 793
2331 AUCUUAGU CUGAUGAG X CGAA AUUUCUUG382 CAAGAAAT A ACTAAGAT 794
2335 UCUGAUCU CUGAUGAG X CGAA AGUUAUUU383 AAATAACT A AGATCAGA 795
2340 UCUGCUCU CUGAUGAG X CGAA AUCUUAGU384 ACTAAGAT C AGAGCAGA 796
2361 UGUGUCUC CUGAUGAG X CGAA AUUUCCUU385 AAGGAAAT A GAGACACA 797
2377 UUUUUGAA CUGAUGAG X CGAA AGUUtJWCT386 AAAAAACT C TTCAAAAA 798
2379 AU<JUUUUG CUGAUGAG X CGAA AGAGUUUU387 AAAACTCT T CAAAAAAT 799
2380 GAUfJUUULT CUGAUGAG X CGAA AAGAGUUU388 AAACTCTT C AAAAAATC 800
2388 GAUUCAUU CUGAUGAG.X CGAA AUUUUUUG389 CAAAAAAT C AATGAATC 801
2396 AGCUCCUG CUGAUGAG X CGAA AUUCAUUG390 CAATGAAT C CAGGAGCT 802
2408 UUUCAAAA CUGAUGAG X CGAA ACCAGCUC391 GAGCTGGT T TTTTGAAA 803
2409 GUUUCAAA CUGAUGAG X CGAA AACCAGCU392 AGCTGGTT T TTTGAAAC 804
2410 CGUUUCAA CUGAUGAG X CGAA AAACCAGC393 GCTGGTTT T TTGAAACG 805
2411 UCGUUUCA CUGAUGAG X CGAA AAAACCAG394 CTGGTTTT T TGAAACGA 806
2412 AUCGUUUC CUGAUGAG X CGAA AAAAACCA395 TGGTTTTT T GAAACGAT 807
2421 AUUUUGUU CUGAUGAG X CGAA AUCGUUUC396 GAAACGAT C AACAAAAT 808
2430 UGUCUAUC CUGAUGAG X CGAA AUUUUGUU397 AACAAAAT T GATAGACA 809
2434 CUAGUGUC CUGAUGAG X CGAA AUCAAUUU398 AAATTGAT A GACACTAG 810
2441 AGUCUUGC CUGAUGAG X CGAA AGUGUCUA399 TAGACACT A GCAAGACT 811
2450 , UUCUUUAU CUGAUGAG X CGAA AGUCUUGC400 GCAAGACT A ATAAAGAA 812
2453 UUCUUCUU CUGAUGAG X CGAA AUUAGUCU401 AGACTAAT A AAGAAGAA 813
2475 UUCUAUUU CUGAUGAG X CGAA AWCUUCU402 AGAAGAAT C AAATAGAA 814
2480 AUUGCUUC CUGAUGAG X CGAA AUUUGAUU403 AATCAAAT A GAAGCAAT 815
2489 UCAUUUUU CUGAUGAG X CGAA AUUGCUUC404 GAAGCAAT A AAAAATGA 816
2499 AUCCCCUU CUGAUGAG X CGAA AUCAUUUU405 AAAATGAT A AAGGGGAT 817
2508 GGUGGUGA CUGAUGAG X CGAA AUCCCCUU406 AAGGGGAT A TCACCACC 818
2510 UUGGUGGU CUGAUGAG X CGAA AUAUCCCC407 GGGGATAT C ACCACCAA 819
2520 UUCUGUGG CUGAUGAG X CGAA AUUGGUGG408 CCACCAAT C CCACAGAA 820
2531 UGGUGGUU CUGAUGAG X CGAA AUUUCUGU409 ACAGAAAT A AACCACCA 821
2541 UAUUCUCU CUGAUGAG X CGAA AUGGUGGU410 ACCACCAT C AGAGAATA 822
2549 GUUUGUAG CUGAUGAG X CGAA AUUCUCUG411 CAGAGAAT A CTACAAAC 823
2552 GGUGUUUG CUGAUGAG X CGAA AGUAUUCU412 AGAATACT A CAAACACC 824
Input Sequence = HSU29607. Cut Site = UH/.
Stem Length = 8 . Core Sequence = CUGAUGAG X CGAA (X = GCCGUUAGGC or other
stem II)
Seq 1 = HSU29607 (Human methionine aminopeptidase mRNA, complete cds., 2569
bp)
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Table 10
Table 10: Human methionine aminopeptidase type 2 (MetAP-2) NCH Ribozyme and
Target Sequence
Nt. Ribozyme Seq. Substrate Seq.
osition Sequence ID Sequence ID
Nos. Nos.
CCCGAGAG CUGAUGAGX IACGAGGG 825 CCCTCGTC T CTCTCGGG1255
CGAA
12 UGCCCGAG CUGAUGAGX IAGACGAG 826 CTCGTCTC T CTCGGGCA1256
CGAA
14 GUUGCCCG CUGAUGAGX IAGAGACG 827 CGTCTCTC T CGGGCAAC1257
CGAA
CGCCAUGU CUGAUGAGX ICCCGAGA 828 TCTCGGGC A ACATGGCG1258
CGAA
23 GCCCGCCA CUGAUGAGX IUUGCCCG 829 CGGGCAAC A TGGCGGGC1259
CGAA
49 CUCCCGGA CUGAUGAGX ICCGCUAC 830 GTAGCGGC C TCCGGGAG1260
CGAA
50 GCUCCCGG CUGAUGAGX IGCCGCUA 831 TAGCGGCC T CCGGGAGC1261
CGAA
52 UGGCUCCC CUGAUGAGX IAGGCCGC 832 GCGGCCTC C GGGAGCCA1262
CGAA
59 AUUCAGGU CUGAUGAGX ICUCCCGG 833 CCGGGAGC C ACCTGAAT1263
CGAA
60 CAUUCAGG CUGAUGAGX 834 CGGGAGCC A CCTGAATG1264
CGAA
IGCUCCCG
62 GCCAUUCA CUGAUGAGX IUGGCUCC 835 GGAGCCAC C TGAATGGC1265
CGAA
63 CGCCAUUC CUGAUGAGX 836 GAGCCACC T GAATGGCG1266
CGAA
IGUGGCUC
74 UGGAUCCA CUGAUGAGX IUCGCCAU 837 ATGGCGAC C TGGATCCA1267
CGAA
75 CUGGAUCC CUGAUGAGX IGUCGCCA 838 TGGCGACC T GGATCCAG1268
CGAA
81 UGUCGUCU CUGAUGAGX IAUCCAGG 839 CCTGGATC C AGACGACA1269
CGAA
82 CUGUCGUC CUGAUGAGX IGAUCCAG 840 CTGGATCC A GACGACAG1270
CGAA
89 UUCUUCCC CUGAUGAGX IUCGUCUG 841 CAGACGAC A GGGAAGAA1271
CGAA
103 GUAGAGGC X ICUCCUUC 842 GAAGGAGC T GCCTCTAC1272
CUGAUGAG CGAA
106 GCCGUAGA CUGAUGAGX ICAGCUCC 843 GGAGCTGC C TCTACGGC1273
CGAA
107 AGCCGUAG CUGAUGAGX IGCAGCUC 844 GAGCTGCC T CTACGGCT1274
CGAA
109 UCAGCCGU CUGAUGAGX IAGGCAGC 845 GCTGCCTC T ACGGCTGA1275
CGAA
115 GCUUCCUC CUGAUGAGX ICCGUAGA 846 TCTACGGC T GAGGAAGC1276
CGAA
124 UUCUUGGC CUGAUGAGX ICUUCCUC 847 GAGGAAGC A GCCAAGAA1277
CGAA
127 UUUUUCUU X 848 GAAGCAGC C AAGAAAAA1278
CUGAUGAG CGAA
ICUGCUUC
128 WUUUIJCU CUGAUGAGX IGCUGCUU 849 AAGCAGCC A AGAAAA.AA1279
CGAA
158 AGGCCCUU X ICUCUUCU 850 AGAAGAGC A AAGGGCCT1280
CUGAUGAG CGAA
165 CUGCAGAA X ICCCUUUG 851 CAAAGGGC C TTCTGCAG1281
CUGAUGAG CGAA
166 GCUGCAGA CUGAUGAGX IGCCCUUU 852 AAAGGGCC T TCTGCAGC1282
CGAA
169 CCUGCUGC CUGAUGAGX IAAGGCCC 853 GGGCCTTC T GCAGCAGG1283
CGAA
172 UCCCCUGC CUGAUGAGX ICAGAAGG 854 CCTTCTGC A GCAGGGGA1284
CGAA
175 UGUUCCCC CUGAUGAGX ICUGCAGA 855 TCTGCAGC A GGGGAACA1285
CGAA
183 CAGGUUCC CUGAUGAGX IUUCCCCU 856 AGGGGAAC A GGAACCTG1286
CGAA
189 CUUUAUCA CUGAUGAGX IUUCCUGU 857 ACAGGAAC C TGATAAAG1287
CGAA
190 UCUUUAUC CUGAUGAGX IGUUCCUG 858 CAGGAACC T GATAAAGA1288
CGAA
202 GAGGCUCC CUGAUGAGX IAUUCUUU 859 AAAGAATC A GGAGCCTC1289
CGAA
208 UCCACUGA CUGAUGAGX ICUCCUGA 860 TCAGGAGC C TCAGTGGA1290
CGAA
209 AUCCACUG CUGAUGAGX IGCUCCUG 861 CAGGAGCC T CAGTGGAT1291
CGAA
211 UCAUCCAC CUGAUGAGX IAGGCUCC 862 GGAGCCTC A GTGGATGA1292
CGAA
226 AACUGUCU CUGAUGAGX ICUACUUC 863 GAAGTAGC A AGACAGTT1293
CGAA
231 UUUCCAAC CUGAUGAGX 864 AGCAAGAC A GTTGGAAA1294
CGAA
IUCUUGCU
244 UCCAAUGC CUGAUGAGX IAUCUUUC 865 GAAAGATC A GCATTGGA1295
CGAA
247 UCUUCCAA X ICUGAUCU 866 AGATCAGC A TTGGAAGA1296
CUGAUGAG CGAA
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 3
~~ TTENANT LES PAGES 1 A 250
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