Note: Descriptions are shown in the official language in which they were submitted.
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ANTISENSE OLIGONUCLEOTIDE CHEMOTHERAPY FOR
BENIGN HYPERPLASIA OR CANCER OF THE PROSTATE
Field of the Invention
The present invention relates to the field of chemotherapy for hyperplasias and cancers
and, in particular, to chemotherapy for benign hyperplasia or cancer of the prostate. In addition,
the invention relates to the field of antisense oligonucleotides and their use in human hyperplasia
and cancer therapy.
Background of the Invention
Treatment of carcinoma of the prostate was one of the first successes of cancer
chemotherapy, using the therapeutic program of castration and/or anti-androgen hormonal
tre~tment~ introduced by Charles Huggins in the 1 940s. A r~m~rk~hle relief of symptoms and
objective regression of bony metastases occurs under this endocrine therapeutic program.
U~ ately, after a "golden period" which lasts roughly 18 months, regrowth of the prostate
cancer cells occurs and, in the later stages of the disease, sensitivity to and repression by anti-
androgen hormonal therapy ceases. The conventional regimen of combined chemotherapeutic
agents also is typically ineffective after the golden period, and a downhill clinical course follows,
t~rmin~ting in death.
A key problem had been the silent onset of cancer of the prostate, with growth beyond its
capsule and metastasis to bone too frequently occurring before the first visit to a physician.
During the last half dozen years, there has been increasing recognition of the importance of early
diagnosis and significant improvements in the available tests. As a consequence of early
diagnosis, detection of prostatic cancer still contained within its capsule has become more
frequent. For this situation, radical prostatectomy has largely supplanted the traditional
castration/estrogen therapy. Radiation targeted to the prostate itself and to any proximal capsular
infiltration has also become a prominent modality of therapy. When these two therapeutic
approaches fail to halt progression of the disease, which is all too often (see, e.g., Gittes ( 1991 );
and Catalona (1994)), the prospect of benefit from available chemotherapy is gloomy.
Less severe but more common than prostatic cancer is benign prostatic hyperplasia
(BPH). This condition may be a precursor to full blown prostatic cancer or may continue for
~l~ç~(les without evolving into the deadly carcinoma. Depending upon the degree of hypertrophy
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and the age of the patient, treatment may range from "watchful waiting" to more aggressive
approaches employing anti-androgen hormonal therapy, transurethral resection, or radical
prostatectomy (see, e.g., Catalona (1994)).
The androgen receptor (AR) binds the male hormone testosterone and, acting at the
5 transcriptional level, regulates the growth of normal prostatic cells. A cDNA for the human AR
was disclosed by T nh~hn et al. (1988). As noted above, anti-androgen or estrogen hormonal
therapy, including physical or chemical castration, may be effective against early stage prostate
cancer but, after a period of roughly 18 months, the patient becomes refractory to the hormonal
therapy. The relapse is believed to be the result of the development or clonal selection of
10 androgen-independent tumor cells in which the AR has mutated or been lost (see, e.g., Taplin, et
al. (1995); Klocker, et al. (1994). Interestingly, in murine androgen-independent prostatic cancer
cells, transfection ~ith an AR cDNA has been shown to inhibit growth in the presence of
testosterone (Suzuki, et al. (1994)).
The acidic fibroblast growth factor (ocFGF), also known as the heparin binding growth
15 factor type one (HBGF- 1), is an androgen-regulated mitogen produced by prostatic cells. An
mRNA sequence for a human allele of aFGF was disclosed in Harris, et al. (1991). Mansson, et
al. (1989) found that o~FGF was expressed in normal imm~tnre rat prostate but not in normal
mature rat prostate. In cancerous rat prostatic cell lines, they found ccFGF expression similar to
that in imm~tllre rat prostate.
Summar~ of the Invention
The present invention provides methods for treating a patient diagnosed as having benign
prostatic hyperplasia or a prostatic cancer. The methods include ~nnini~t~ring to the patient a
therapeutically effective amount of a composition comprising an antisense oligonucleotide which
25 selectively hybridizes to an AR or c~FGF gene or mRNA sequence of the patient, thereby
inhibiting the expression of the AR or ocFGF gene or mRNA sequence. This inhibition of the AR
or ocFGF genes or mRNAs by antisense oligonucleotides results in a significant inhibition of the
growth or survival of prostatic cells. As a result, the methods provide a useful new means of
treating benign prostatic hyperplasia and prostatic cancer. The methods are particularly useful in
30 treating prostate cancer patients who have become refractory to anti-androgen hormonal therapy.
The AR antisense oligonucleotides may comprise at least 10 consecutive bases from SEQ
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ID NO.: 1, at least 10 consecutive bases from a genomic sequence corresponding to SEQ ID
NO.: 1, or oligonucleotides that hybridize to the complements of these sequences under
physiological conditions. More preferably, the antisense oligonucleotides comprise at least 15
consecutive bases, and most preferably, 20-30 consecutive bases from the above-described
S sequences.
The aFGF antisense oligonucleotides may comprise at least 10 consecutive bases from
any one of SEQ ID NO.: 2, SEQ ID NO.: 3 or SEQ ID NO.: 4, at least 10 consecutive bases from
the joined exons of SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4, or oligonucleotides that
hybridize to the complements of these sequences under physiological conditions. More
10 preferably, the antisense oligonucleotides comprise at least 15 consecutive bases, and most
preferably, 20-30 consecutive bases from the above-described sequences.
Examples of sequences of the invention include, but are not limited to, those disclosed as
SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, and SEQ ID NO.: 8.
In ~lt;r~ ed embo-liment.~, all of the above-described oligonucleotides are modified
15 oligonucleotides. In one set of embo-limentc, the modified oligonucleotide includes at least one
synthetic intermlcleoside linkage such as a phosphorothioate, alkylphosphonate,
phosphorodithioate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate,
carbonate, phosphate triester, acetamidate, or carboxymethyl ester.
In other embodiments with modified oligonucleotides, the modified oligonucleotide has
20 at least one low molecular weight organic group covalently bound to a phosphate group of said
oligonucleotide. In another set of embodiments, the modified oligonucleotide has at least one
low molecular weight organic group covalently bound to a 2' position of a ribose of said
oligonucleotide. Such low molecular weight organic groups include lower alkyl chains or
aliphatic groups (e.g., methyl, ethyl, propyl, butyl), substituted alkyl and aliphatic groups (e.g.,
25 aminoethyl, aminopropyl, aminohydroxyethyl, aminohydroxypropyl), small saccharides or
glycosyl groups.
In another set of embo-1iment~ the modified oligonucleotide has covalently attached
thereto a prostate-targeting compound such as an androgen, androgen derivative, estrogen,
estrogen derivative, estr~mll~tine, emcyt or estracyt.
In pl~r~lled embodiments, the ~nti~en~e oligonucleotides are :~mini~tered intravenously
at a dosage between 1.0 ~Lg and 100 mg per kg body weight of the patient.
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The present invention also provides for any or all of the above-described antisense
oligonucleotides, including the various modified oligonucleotides, in a ph~rmslreutical
composition. The antisense oligonucleotides are admixed with a sterile ph~nn:~relltically
acceptable carrier in a therapeutically effective amount such that the isolated ~nti~t-n~e
5 oligonucleotide selectively hybridizes to the AR or ~FGF gene or mRNA sequence when
~rlmini.stered to a patient. A pharmaceutical kit is also provided in which such a ph~rm~eeutical
composition is combined with a ph~rrn~reutically acceptable carrier for intravenous
s~1mini~tration.
The methods and products of the present invention further include ~nti~çn~e
10 oligonucleotides, as described above, directed at a PSA gene, a probasin gene, an estrogen
receptor gene, a telomerase gene, a prohibitin gene, a src gene, a ras gene, a myc gene, a blc-2
gene, a protein kinase-A gene, a plasminogen activator urokinase gene and a methyl transferase
gene.
Detailed De~el ;I lion of the Invention
The present invention provides new methods for the tre~tment of cancer of the prostate
and pharmaceutical compositions useful therefor. It is now disclosed that antisense
oligonucleotides complementary to genes which are expressed predomin~ntly or strongly in
prostatic cells are effective for inhibiting the growth of and/or killing hyperplastic or cancerous
20 cells of prostatic origin. In particular, the present invention provides oligonucleotides, including
modified oligonucleotides, which have antisense homology to a sufficient portion of either the
AR or c~FGF gene such that they inhibit the t;x~lession of that gene. Surprisingly, inhibition of
either of these genes, even in androgen-resistant prostatic cancer cells, inhibits the growth of
these cells. Because the ~nti~n~e oligonucleotides ofthe invention can be ~lmini~tered
25 systemically but selectively inhibit prostate cells, the present invention has particular utility in
late stage prostate cancer which has metastasized.
Definitions
In order to describe more clearly and concisely the subject matter of the present
invention, the following definitions are provided for specific terms used in the claims appended
30 hereto:
AR. As used herein, the abbreviation "AR" refers to the androgen receptor well known
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s
in the art and described in the various references cited herein. A cDNA sequence of the hurnan
AR gene was disclosed in Lubahn et al. (1988). The Lubahn et al. (1988)sequence is available
on GenBank (Accession nurnber J03180) and is reproduced here as SEQ. ID NO.: 1. The
translation initiation codon of this gene is found at base positions 363-365 and the stop codon is
5 at positions 3120-3122 of SEQ ID NO.: 1. As will be obvious to one of ordinary skill in the art,
other alleles of the AR gene, including other human alleles and homologues from other
m~mm~ n species, encoding an AR protein and hybridizing to SEQ ID NO.: 1 under stringent
hybridization conditions, will exist in natural populations and are embraced by the term "AR
gene" as used herein.
~aFGF. As used herein, the term "aFGF" refers to the aFGF protein known in the art and
described in the various references cited herein. The genomic DNA of one allele of the human
aFGF gene has been partially sequenced and was disclosed in Wang et al. (1989). The Wang et
al.(l 989) sequences cover the three exons of the aFGF gene as well as some 5', 3' and intron
sequences. These sequences are available on GenBank (Accession numbers M23017, M23086
15 and M23087) and are reproduced here as SEQ. ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4.
A partial cDNA sequence for a human aFGF gene also may be found in Harris et al. (1991). The
locations of the exons are located in the sequence listings. The translation initiation codon is
found at positions 602-604 of SEQ ID NO.: 2 and the stop codon is found at positions 496-498.
In addition, as will be obvious to one of ordinary skill in the art, other alleles of the c~FGF gene,
20 including other human alleles and homologues from other m~mm~ n species, encoding an
aFGF protein and hybridizing to one or more of SEQ ID NO.: 2, SEQ ID NO.: 3 or SEQ ID
NO.: 4 under stringent hybridization conditions, will exist in natural populations and are
embraced by the terrn "aFGF gene" as used herein.
.~nti~e~e Oli~onucleotides. As used herein, the term "antisense oligonucleotide" or
25 "~nti~n~e" describes an oligonucleotide that is an oligoribonucleotide,
oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide
which hybridizes under physiological conditions to DNA comprising a particular gene or to an
mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the
translation of that mRNA. In particular, by an "AR-antisense oligonucleotide" and bv an
~ 30 "aFGF-antisense oligonucleotide" are meant oligonucleotides which hybridize under
physiological conditions to the AR gene/mRNA or aFGF gene/mRNA and, thereby. inhibit
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transcription/translation of the AR and aFGF genes/mRNAs, respectively. The antisense
molecules are designed so as to h~ r~L~; with transcription or translation of AR or aFGF upon
hybridization with the target. Those skilled in the art will recognize that the exact length of the
antisense oligonucleotide and its degree of complementarity will depend upon the specific target
S selected, including the sequence of the target and the particular bases which comprise that
sequence. It is preferred that the antisense oligonucleotide be selected so as to hybridize
selectively with the target under physiological conditions, i.e., to hybridize substantially more to
the target sequence than to any other sequence in the target cell under physiological conditions.
Strin~e~t hvbridization conditions. As used herein, the term "stringent hybridization
10 conditions" means hybridization conditions from 30~C-60~C and from 5x to O.lx SSC. Highly
stringent hybridization conditions are at 45~C and O.lx SSC. "Stringent hybridization
conditions" is a term of art understood by those of ordinary skill in the art. For any given nucleic
acid sequence, stringent hybridization conditions are those conditions of temperature and buffer
solution which will permit hybridization of that nucleic acid sequence to its complementary
15 sequence and not to substantially dirr~lcllL sequences. The exact conditions which constitute
"stringent" conditions, depend upon the length of the nucleic acid sequence and the frequency of
occurrence of subsets of that sequence within other non-identical sequences. By varying
hybridization conditions from a level of stringency at which no hybridization occurs to a level at
which hybridization is first observed, one of ordinary skill in the art can, without undue
20 e~ hllentation, determine conditions which will allow a given sequence to hybridize only with
identical sequences. Suitable ranges of such stringency conditions are described in Krause,
M.H.. and S.A. Aaronson, Methods in Enz,vmolo~y. 200:546-556 (1991). As used herein with
respect to in vivo hybridization conditions, the term "physiological conditions" is considered
functionally equivalent to the in vitro stringent hybridization conditions.
25 I Des;~n of AR and aFGF Anti~ n~e Oli~onucleotides
The present invention depends, in part, upon the discovery that the selective inhibition of
the expression of AR or aFGF by antisense oligonucleotides in prostatic cells effectively inhibits
cell growth and/or causes cell death.
Based upon SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4, or
30 upon allelic or homologous genomic or cDNA sequences, one of skill in the art can easily choose
and synth~ci7~ any of a number of al)plopl;ate antisense molecules for use in accordance with the
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present invention. In order to be sufficiently selective and potent for AR or aFGF inhibition,
such ~3ntieçnee oligonucleotides should comprise at least 10 and, more preferably, at least 15
consecutive bases which are complementary to the AR or aFGF mRNA transcripts. Most
preferably, the ~ntieçnee oligonucleotides comprise a complementary sequence of 20-30 bases.
5 Although oligonucleotides may be chosen which are ~ntieçnee to any region of the AR or aFGF
genes or mRNA transcripts, in preferred embo(1imente the ~ntiei nee oligonucleotides correspond
to N-tçrminsll or 5' upstream sites such as kanslation initiation, transcription initiation or
promoter sites. In addition, 3'-untr:~nel~t~-l regions or telomerase sites may be targeted.
Targeting to mRNA splicing sites has also been used in the art but may be less preferred if
10 alternative mRNA splicing occurs. In addition, the AR or aFGF ~ntie~nee is, preferably, targeted
to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al. (1994)) and at
which proteins are not expected to bind. Finally, although, SEQ ID NO.: 1 discloses a cDNA
sequence and SEQ ID NO.: 2, SEQ ID NO.:3 and SEQ ID NO.: 4 disclose genomic DNA
sequences, one of ordinary skill in the art may easily derive the genomic DNA corresponding to
15 the cDNA of SEQ ID NO.: 1 and may easily obtain the cDNA sequence corresponding to SEQ
ID NO.: 2, SEQ ID NO.:3 and SEQ ID NO.: 4. Thus, the present invention also provides for
~ntiecnee oligonucleotides which are complementary to the genomic DNA corresponding to SEQ
ID NO.: 1 and the cDNA corresponding to SEQ ID NO.: 2, SEQ ID NO.: 3 and SEQ ID NO.: 4.
Similarly, ~ntieçnee to allelic or homologous cDNAs and genomic DNAs are enabled without
20 undue ~ hnentation.
As will be understood by one of ordinary skill in the art, the antisense oligonucleotides of
the present invention need not be perfectly complementary to the AR or aFGF genes or mRNA
transcripts in order to be effective. Rather, some degree of miem~tches will be acceptable if the
slntieenee oligonucleotide is of sufficient length. In all cases, however, the oligonucleotides
25 should have sufficient length and complementarity so as to hybridize to an AR or ocFGF
transcript under physiological conditions. Preferably, of course, mi~m~trhes are absent or
minim~l In addition, although it is not recommended, the antisense oligonucleotides may have
one or more non-complementary sequences of bases inserted into an otherwise complementary
antisense oligonucleotide sequence. Such non-complementary sequences may "loop" out of a
30 duplex formed by an AR or aFGF transcript and the bases fl~nkin~ the non-complementary
region. Therefore, the entire oligonucleotide may retain an inhibitory effect despite an
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apparently low percentage of complementarity. Of particular importance in this respect is the
use of self-stabilized or hairpin oligonucleotides. Such oligonucleotides, or modified
oligonucleotides, have a sequence at the 5' and/or 3' end which is capable of folding over and
forming a duplex with itself. The duplex region, which is preferably at least 4-6 bases joined by
5 a loop of 3-6 bases, stabilizes the oligonucleotide against degradation. These self-stabilized
oligonucleotides are easily designed by adding the inverted complement of a 5' or 3' AR or o~FGF
sequence to the end of the oligonucleotide (see, e.g., Table 1, SEQ ID NO.: 6 and SEQ ID NO.:
7, Tang, J.-Y., et al. (1993) Nucleic Acids Res. 21:2729-2735).
In one set of embodiments, the AR and ~FGF ~nti~en.~e oligonucleotides of the invention
10 may be composed of"natural" deoxyribonucleotides, ribonucleotides, or any combination
thereof. That is, the 5' end of one nucleotide and the 3' end of another nucleotide may be
covalently linked, as in natural systems, via a phosphodiester internucleoside linkage. These
oligonucleotides may be prepared by art recognized methods which may be ca~ied out manually
or by an automated synthesizer.
In preferred embo~liment~, however, the antisense oligonucleotides of the invention also
may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a
number of ways which do not prevent them from hybridizing to their target but which enhance
their stability or targeting to prostatic cells or which otherwise enhance their therapeutic
effectiveness. The term "modified oligonucleotide" as used herein describes an oligonucleotide
20 in which (1) at least two of its nucleotides are covalently linked via a synthetic intf?n~llcleoside
linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide
and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with
nucleic acids has been covalently attached to the oligonucleotide.
Preferred synthetic int~ rmlcleoside linkages are phosphorothioates, aLkylphosphonates,
25 phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,
carbonates, phosphate triesters, acetamidate, and carboxymethyl esters. Further, one or more of
the 5' ~ 3' phosphate group may be covalently joined to a low molecular weight (e.g., 15-500 Da)
organic group. Such low molecular weight organic groups include lower alkyl chains or
aliphatic groups (e.g., methyl, ethyl, propyl, butyl), substituted alkyl and aliphatic groups (e.g.,
30 arninoethyl, aminopropyl, aminohydroxyethyl, aminohydroxypropyl), small saccharides or
glycosyl groups. Other low molecular weight organic modifications include additions to the
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int~rmlrleoside phosphate linkages such as cholesteryl or diamine compounds with varying
numbers of carbon residues between the amino groups and t~rmin~l ribose. Oligonucleotides
with these linkages or other modifications can be prepared according to known methods (see,
e.g., Agrawal and Goodchild (1987); Agrawal et al. (1988); Uhlmann et al. (1990); Agrawal et
S al. (1992); Agrawal (1993); and U.S. Pat. No. 5,149,798).
The term "modified oligonucleotide" also encompasses oligonucleotides with a
covalently modified base and/or sugar. For example, modified oligonucleotides include
oligonucleotides having backbone sugars which are covalently attached to low molecular weight
organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at
10 the 5' position. Thus modified oligonucleotides may include a 2'-O-alkylated ribose group such
as a 2'-O-methylated ribose. In addition, modified oligonucleotides may include sugars such as
arabinose instead of ribose. ~ltern~tively, the modified oligonucleotides may be branched
oligonucleotides. Unoxidized or partially oxidized oligonucleotides having a substitution in one
or more nonbridging oxygen per nucleotide in the molecule are also considered to be modified
15 oligonucleotides.
Also considered as modified oligonucleotides are oligonucleotides having prostate-
targeting, nuclease resistance-conferring, or other bulky substituents and/or various other
structural modifications not found in vivo without hurnan intervention. The androgen receptor
and other hormonal receptor sites on prostate cells allow for targeting antisense oligonucleotides
20 specifically or particularly to prostatic cells. Attachment of the antisense oligonucleotides by a
molecular "tether" (e.g., an alkyl chain) to estrz~n~ tine, emcyt or estracyt (Sheridan and Tew
(1991)), for example, may provide prostatic targeting and the possibility of covalent alkylation of
host prostatic DNA. Estramustine targets particularly to the ventral prostate (Forsgren, et al.
(1979)). Similarly, one may covalently attach androgen, estrogen, androgen or estrogen
25 der*atives, or other prostate cell ligands to ~nti~ence oligonucleotides using tethers and
conjugating linkages for prostatic targeting. Finally, one may of course covalently attach other
chemotherapeutic agents (e.g., dexamethasone, vinblastine, etoposide) to the antisense
oligonucleotides for enhanced effect.
The most plc~r~lled modified oligonucleotides are hybrid or chimeric oligonucleotides in
30 which some but not all of the phosphodiester linkages, bases or sugars have been modified.
Hybrid modified ~nti~cn~e oligonucleotides may be composed, for example, of stretches of ten
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2'-O-alkyl nucleotides or ten phosphorothioate synthetic linkages at the S' and/or 3' ends, and a
segment of seven unmodified oligodeoxynucleotides in the center, or of similar terrninal
segment~ of alkyl phosphonates, with central P=S or P=O oligonucleotides (Agrawal, et al.
(1990); Metelev, et al. (1994)). The currently most preferred modified oligonucleotides are 2'-O-
5 methylated hybrid oligonucleotides. Since degradation occurs mainly at the 3' end, secondarilyat the 5' end, and less in the middle, unmodified oligonucleotides located at this position can
acJiv~te Rl~a~e H, ~nd yet arç degraded slowly. F~hermorç, the T~n of suçh a 27-mer is
approximately 20~C higher than that of a 27-mer all phosphorothioate oligodeoxynucleotide.
This greater affinity for the targeted genomic area can result in greater inhibiting efficacy.
10 Obviously, the nurnber of synthetic linkages at the termini need not be ten and synthetic linkages
may be combined with other modifications, such as alkylation of a 5' or 3' phosphate, or 2'-O-
alkylation. Thus, merely as another example, one may produce a modified oligonucleotide with
the following structure, where B represents any base, R is an alkyl, aliphatic or other substituent,
the subscript S represents a synthetic (e.g. phosphorothioate) linkage, and each n is an
15 independently chosen integer from 1 to about 20:
OH
s'(BS)nBBBB- - ~BBBB(Bs)nB - P=O3'
O--R
II. Products and Methods of Tr~tment for BPH and Prostate Cancer
The methods of the present invention represent new and useful additions to the field of
benign prostate hyperplasia or prostate cancer therapy. In particular, the methods of the present
25 invention are especially useful for late stage prostate cancer in which metastases have occurred
and in which the cells have become resistant to estrogen or anti-androgen therapy. The methods
may, however, also be used in benign prostate hyperplasia or early stage prostate cancer and may
provide a snkstit~lte for more radical procedures such as transurethral resection, radical
prostatectomy, or physical or chemical castration. The products of the present invention include
30 the isolated anti~n~e oligonucleotides described above. As used herein, the term "isolated" as
applied to an anti~n~e oligonucleotide means not covalently bound to and physically separated
from the S' and 3' sequences which flank the corresponding antisense sequence in nature.
Atlmini~tration of the AR or o~FGF ~nti~çn~e oligonucleotides may be oral, intravenous,
77454.1
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parenteral, cutaneous or subcutaneous. For BPH or when the site of a prostatic tumor is known,
the ~mini.~tration also may be localized to the prostate or to the region of the tumor by injection
to or perfusion of the site.
AR or aFGF antisense oligonucleotides may be ~(lmini~tered as part of a pharrnaceutical
5 composition. Such a ph~rm~reutical composition may include the antisense oligonucleotides in
combination with any standard physiologically and/or ph~rm~reutically acceptable carriers
which are known in the art. The compositions should be sterile and contain a therapeutically
effective amount of the ~nti.~f n~e oligonucleotides in a unit of weight or volume suitable for
~1mini~tration to a patient. The term "ph~rm~relltically acceptable" means a non-toxic material
10 that does not interfere with the effectiveness of the biological activity of the active ingredients.
The term "physiologically acceptable" refers to a non-toxic m~t~ri~l that is compatible with a
biological system such as a cell, cell culture, tissue, or org~ni~m. The characteristics of the
carrier will depend on the route of ~-1mini~tration. Physiologically and ph~rm~ceutically
acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other
15 m~t~ri~l~ which are well known in the art. The pharmaceutical composition of the invention may
also contain other active factors and/or agents which inhibit prostate cell growth or increase cell
death. Such additional factors and/or agents may be included in the phzlrm~ eutical composition
to produce a synergistic effect or to minimi7f side-effects caused.
The pharrnaceutical composition of the invention may be in the forrn of a liposome in
20 which the AR or aFGF ~nti~çn~e oligonucleotides are combined, in addition to other
ph~rm~eutically acceptable carriers, with amphipathic agents such as lipids which exist in
aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers which are
in aqueous solution. Suitable lipids for liposomal forrnulation include, without limitation,
monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the
25 like. P~ udlion of such liposomal formulations is within the level of skill in the art, as
disclosed, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No.
4,837,028; and U.S. Pat. No. 4,737,323.
The ph~rmzlreutical composition of the invention may further include compounds such as
cyclodextrins and the like which enhance delivery of oligonucleotides into cells. When the
30 composition is not al1mini~tered systemically but, rather, is injected at the site of the target cells,
cationic detergents (e.g. Lipofectin) may be added to enhance uptake.
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12
When a therapeutically effective amount of AR or o~FGF antisense oligonucleotides is
~lmini~tered orally, the oligonucleotides will be in the form of a tablet, capsule, powder, solution
or elixir. When ~fimini~tered in tablet form, the pharmaceutical composition of the invention
may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and
5 powder may contain from about 5 to 95% of the AR and/or aFGF antisense oligonucleotides and
preferably from about 25 to 90% of the oligonucleotides. When ~lmini~tered in liquid form, a
liquid carrier such as water, pekoleum, oils of animal or plant origin such as peanut oil, mineral
oil, soybean oil, sesame oil, or synthetic oils may be added. The liquid form of the
I)h~rmzlreutical composition may further contain physiological saline solution, dexkose or other
10 saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
When ~lmini~trred in liquid form, the ph~rm~reutical composition may contain from about 0.5
to 90% by weight of an AR and/or aFGF antisense oligonucleotide and preferably from about 1
to 50% ofthe oligonucleotide.
When a therapeutically effective amount of an AR or o~FGF antisense oligonucleotide is
15 ~r1mini~trred by inkavenous, cutaneous or subcutaneous injection, the oligonucleotides will be in
the form of a pyrogen-free, parenterally acceptable aqueous solution. The p~ ~dlion of such
parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is
within the skill in the art. A preferred pharrnaceutical composition for intravenous, cutaneous, or
subcutaneous injection should contain, in addition to the ~nti~en~e oligonucleotides, an isotonic
vehicle such as Sodium Chloride Injection, Ringer's Injection, Dexkose Injection, Dextrose and
Sodium Chloride Injection, Lactated Ringer's Injection, or another vehicle as known in the art.
l~he ph~rrn~reutical composition of the present invention may also contain stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
In ~ler~ d embodiments, when the target cells are readily accessible, ~-1mini~tration of
the ~nti~çn~e oligonucleotides is localized to the region of the targeted cells in order to m~lcimi71
the delivery of the ~nti~en~e and to minimi7P the amount of antisense needed per treatment.
Thus, in one preferred embodiment, ~flmini~kation is by direct injection at or perfusion of the
site of the targeted cells, such as a tumor. Alternatively, the ~nti~.on~e oligonucleotides may be
adhered to small particles (e.g., microscopic gold beads) which are impelled through the
30 membranes of the target cells (see, e.g., U.S. Pat. No. 5,149,655).
In another series of embodiments, a recombinant gene is constructed which encodes an
CA 02239976 l998-03-l7
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13
AR or aFGF antisense oligonucleotide and this gene is introduced within the targeted cells on a
vector. Such an AR or aFGF antisense gene may, for example, consist of the normal AR or
aFGF sequence, or a subset of the normal sequences, operably joined in reverse orientation to a
promoter region. An operable antisense gene may be introduced on an integration vector or may
5 be introduced on an expression vector. In order to be most effective, it is preferred that the
- antisense sequences be operably joined to a strong eukaryotic promoter which is inducible or
con~liLuliv~ly ~ essed.
In all of the above-described methods of treatment, the AR and/or aFGF antisenseoligonucleotides are ~mini~t~red in therapeutically effective amounts. As used herein, the term
lO "therapeutically effective amount" means that amount of antisense which, under the conditions of
a~lmini~tration, including mode of ~lmini~tration and presence of other active components, is
sufficient to result in a me~ningful patient benefit, i.e., the killing or inhibition of the gro~,-vth of
target cells.
The amount of AR and/or aFGF antisense oligonucleotides in the ph~rm~(~eutical
15 composition of the present invention will depend not only upon the potency of the :~nti~çn~e but
also upon the nature and severity of the condition being treated, and on the nature of prior
tre~tments which the patient has undergone. Ultimately, the attending physician will decide the
amount of ~nti~çn~e with which to treat each individual patient. Initially, the ~tt~-ntiing physician
will ~t1mini~tcr low doses ofthe inhibitor and observe the patient's response. Larger doses of
20 antisense may be ~lmini~tered until the optimal therapeutic effect is obtained for the patient, and
at that point the dosage is not increased further. In preferred embodiments, it is contemplated
that the various rh~rm~t~e~ltical compositions used to practice the method of the present
invention should contain about 1.0 llg to about 100 mg of oligonucleotide per kg body weight.
The duration of intravenous therapy using the ph~rm~eutical compositions of the present
25 invention will vary, depending on the severity of the disease being treated and the condition and
potential idiosyncratic response of each individual patient. Because a bolus of oligonucleotides,
particularly highly negatively-charged phosphorothioate modified oligonucleotides, may have
adverse side effects (e.g., rapid lowering of blood pressure), slow intravenous ~1mini~tration is
preferred. Thus, intravenous ;~mini~tration of therapeutically effective amounts over a 12-24
30 hour period are contemplated. Ultimately the attending physician will decide on the ap~ liate
duration of intravenous therapy using the pharmaceutical composition of the present invention.
77454.1
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14
The following examples of the use of AR and ocFGF antisense are presented merely to
illustrate some of the oligonucleotides, including modified oligonucleotides, that may be
employed according to the present invention. The particular oligonucleotides used, therefore,
should not be conskued as limiting of the invention but, rather, as indicative of the wide range of
5 oligonucleotides which may be employed. As will be obvious to one of ordinary skill in the art
in light of the present disclosure, a great many equivalents to the presently disclosed ~nti~n~e
oligonucleotides and disclosed methods are now available. In particular, other ~nti~en~e
oligonucleotides subst~nti~lly complementary to subsets of SEQ ID NO.: 1, SEQ ID NO.: 2,
SEQ ID NO.: 3 or SEQ ID NO.: 4 and chemical modifications of the same which do not prevent
10 hybridization under physiological conditions, are contemplated as equivalents of the examples
presented below. In general, the use of prostate specific antisense oligonucleotides is
contemplated as a method of selectively inhibiting the growth of or killing prostatic cells. In
particular, the use of antisense oligonucleotides to the eskogen receptor, PSA, probasin,
telomerase, prohibitin, src, ras, myc, blc-2, protein kinase-A, plasminogenctivator urokinase and
15 methyl kansferase genes is contemplated for the tre~ttnent of benign prostatic hyperplasia or
prostatic cancer.
Experimental Exa~nples
The PC3- 1435 permanent cell line of human prostatic cancer, obtained from the
20 American Type Culture Collection, was grown in monolayer culture: The PC3-1435 cells are
from an osseous metastasis and are androgen-insensitive. Cells were grown in Dulbecco's
medium supplemented with 10 percent fetal calf serum, gl~lt~m:~te, pyruvate, penicillin and
skeptomycin, in 25-150 cm flasks, incubated at 37~C in 6 percent CO2-air.
A number of AR and o~FGF antisense oligonucleotides were tested for their inhibitory
25 effect on prostatic cells. The base sequences of these oligonucleotides are disclosed as SEQ ID
NO.: 5 through SEQ ID NO.: 8. SEQ ID NO.: 5 is antisense to positions 927-953 of the AR
gene (SEQ ID NO.: 1). SEQ ID NO.: 6 is a self-stabilized or hairpin oligonucleotide. The first
21 bases are complementary to positions 916-936 of the AR gene. The rem~ining eight are
identical to positions 920-927 of the gene, allowing formation of a 3' hairpin. SEQ ID NO.: 7 is
30 another self-stabilized antisense oligonucleotide. The first 21 bases of this oligonucleotide are
complementary to positions 927-947 of the AR gene. The rem~inin~ eight are identical to
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positions 931 -938 of the gene, allowing for formation of a 3' hairpin. Finally, SEQ ID NO.: 8 is
an ~ntic~n~e sequence corresponding to positions 611-635 of the oLFGF gene.
Table 1 shows some ofthe ~nti~t?n~e oligonucleotides tested. The numbers at the left of
each sequence correspond to the sequence numbers in the sequence listing. ~nti~en~e
oligonucleotides with unmodified or natural intern~lcleoside linkages (P=O) and oligonucleotides
with all phosphorothioate synthetic linkages (P=S) were tested. In addition, modified
oligonucleotides were tested in which just the termin~l two phosphodiester linkages at each end
had been replaced by phosphorothioate synthetic linkages (shown as a subscript S between
nucleotides in Table 1) and/or in which small organic chemical groups (e.g., 2-hydroxy-3-amino-
10 propyl, propylamine) were added to the 3' termin~l phosphate or the penllltim~te 3' phosphate.
Growth of the PC3-1435 cell line in tissue culture monolayers was con~i~tently inhibited
by addition of phosphorothioate-modified oligodeoxynucleotides t~rgeted against the AR or
o~FGF genes and incubation for 24-48 hours thereafter. As the concentration of modified
oligonucleotides is decreased from the 10-20 ~lM level, most effective inhibition occurs with
15 specific ~nti~n~e oligodeoxynucleotides at the 2-5 ~LM level, as contrasted with mi~m~tched
oligodeoxynucleotides (see Tables 2 and 3).
While the effects on cell growth (i.e. cell numbers) are readily manifest, visual substage
microscopy of wells revealed additional features of the inhibition events using AR antisense
oligonucleotides against PC3-1435 cells. The first evidence of antisense inhibition is rupture of
20 the monolayer fabric. The stellate cells in a confluent culture lose contact with their neighbors,
round up individually or in clumps, become pyknotic, and cease growing, as e~mined on
successive days. There is an early loss of adhesiveness to the floor of the plastic wells. These
changes are more severe (see Table 4) than those measured by 3H-thymidine incorporation into
DNA, in other words more drastic than the i",pai""ent of DNA synthesis.
Each of the above-mentioned references and patents are incorporated by reference.
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16
TABLE 1
Antisense Oligonucleotides
Sequence Target
#5 5'CTG-CTG-CTG-TTG-CTG-AAG-GAG-TTG-CAT3 Androgen
receptor,
P=S
#5 5'CTG-CTG-CTG-TTG-CTG-AAG-GAG-TTG-CAT3 Androgen
10 receptor,
P=O
#5 5 ' CSTsG-CTG-CTG-TTG-CTG-AAG-GAG-TTG-CsAsT3' Androgen
receptor,
P=S termini
lS #5 5 ' CTG-CTG-CTG-TTG-CTG-AAG-GAG-TTG-CAT3 Androgen
receptor,
¦ modi~ied with
O organic group
+
H3N-CH2cHcH2O-pl=o
OH OH
O Androgen
25 receptor,
ll modi~ied with
#5 5'CTG-CTG-CTG-TTG-CTG-AAG-GAG-TTG-CA-o-P-o-T3 organic group
CH3CH2CH2NH
#6 5 ' GGA-GTT-GCA-TGG-TGC-TGG-CCT-CAG-CAC-CA3' Androgen
receptor
3' hairpin, P=S
#7 5 ' CTG-TTG-CTG-AAG-GAG-TTG-CAT-AAC-TCC-TT3' Androgen
receptor
3' hairpin, P=S
#8 5 GGG-CTG-TGA-AGG-TGG-TGA-TTT-CCC-C3' . ~FGF, P=S
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17
#85 ' GGG-CTG-TGA-AGG-TGG-TGA-TTT-CCC- C3 ' o~FGF, P=O
S TABLE 2
3H-thymidine incorporation into DNA PC3-1435
human prostate cancer tissue culture
10 Genes Tar~eted Concentration (,uM) CPM~ % inhibition
Control (no oligo) -- 38,000 0
Androgen receptor, (P = S) 2015,000 60
20,000 48
Androgen receptor, (P = S)~ 2010,200 68
24,000 25
l~i~mzlt~h (P = S) 2020,000 47
s 27,000 30
Averages of 3 separate wells
* 3' phosphate modified with -CH2CHOHCHNH3+
TABLE 3
Degree of inhibition of DNA synthesis
in PC3-1435 prostate cancer tissue cultures
Genes targeted Concentration (,uM) CPM ~ % inhibition
Control (no oligo) -- 14,700 0
ccFGF (P=S) 20 2,485 83
4,500 69
Mismatch 20 6,990 51
s 10,750 27
35 ~ Averages of 3 separate wells.
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18
TABLE 4
Morphological Comparison of Treated and Control Cells
Concentration IlM
Gene Tar~et 20 10 5 2
o~FGF ~P=S) 4+ 4+ 1-1/2+ 1+
Androgen receptor (P=S) 3+ 3+ 1+ 1+
Mismatch (P=S) 1-1/2+ 1/2+ 0 0
Observation 24 hours after oligonucleotide addition. Damage: 4+ dev~.~t~1;ng; 3+ severe, 2+
serious; l+ visible; 1/2+ slight; 0 none
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19
References
Agrawal (ed.) Meth. Mol. Biol.~ HumanaPress, Totowa, NJ (1993) Vol. 20.
Agrawal and Goodchild (1987) Tetrahedron Lett. 28:3539-3542.
S Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083.
Agrawal et al.(l990) Proc. Natl. Acad. Sci. (~JSA) 87: 1401-1405.
Agrawal et al. (1992) Trends Biotechnol.10:152-158.
Catalona(1994)N.E. J. Med. 331:996-1004.
Forsgren et al. (1979) CaIlcer Res. 39:5155-5164.
10 Gittes (1991) N,F. J. Med. 324: 236-245.
Harris et al. (1991) in Mol. and Cell. Biol. of Prostate Cancer~ Karr et al (eds.) Plenum Press,
NY,
pp. 315-330.
Klocker, et al. (1994) The Prostate 25:266-273.
15 Lubahn et al. (1988) Mol. F,ntlocr;nl l. 2(12):1265-1275.
Mansson, et al. (1989) Cancer Res. 49:2485-2494.
Metelev et al. (1994) Bioorg,. Medicinal Chem. T~ett~ 4: 2929-2934.
Sainio et al. (1994) Cell. Mol. Neurobiol 14(5):439-457.
Sheridan and Tew (1991) C~ncer Surveys 11 :239-254.
20 Suzuki, et al. (1994) The Prostate 25:310-319.
Taplin et al. (1995) N.E.J. Med. 332(21):1393-1398.
Uhlmann et al. (1990) Chem. Rev. 90:534-583
Wang et al. (1979) Invest. Urol. 17:159-163.
Wang et al. (1989) Mol. Cell. Biol. 9(6):2387-2395.
CA 02239976 1998-03-17
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: WORCESTER FOUNDATION FOR BIOMEDICAL RESEARCH, INC.
(ii) TITLE OF INVENTION: ANTISENSE OLIGONUCLEOTIDE CHEMOTHERAPY
FOR BENIGN HYPERPLASIA OR CANCER OF THE PROSTATE
(iii) NUMBER OF SEQUENCES: 8
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: WOLF, GR~N~lhLD & SACKS, P.C.
(B) STREET: 600 ATLANTIC AVENUE
(C) CITY: BOSTON
(D) STATE: MA
(E) COUNTRY: USA
(F) ZIP: 02210
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l.0, Version #l.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
CA 02239976 l998-03-l7
WO97/11170 PCT~S96/15081
21
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: TWOMEY, MICHAEL ~
(B) REGISTRATION NUMBER: 38,349
(C) REFERENCE/DOCKET NUMBER: W0461/7035
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617-720-3500
(B) TELEFAX: 617-720-2441
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3569 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: l 1n~
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 363.. 3122
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TAATAACTCA GTTCTTATTT GCACCTACTT CAGTGGACAC TGAATTTGGA AGGTGGAGGA 60
CA 02239976 1998-03-17
WO97/11170 PCTAJS96/15081
22
~L'l"l"l'~'l"l"l"L'l' TTCTTTTAAG ATCTGGGCAT ~'~ "l'~AATC TACCCTTCAA GTATTAAGAG 120
ACAGACTGTG AGCCTAGCAG GGCAGATCTT GTCCACCGTG TGTCTTCTTC TGCACGAGAC l80
TTTGAGGCTG TCAGAGCGCT TTTTGCGTGG TTGCTCCCGC AAGTTTCCTT CTCTGGAGCT 240
TCCCGCAGGT GGGCAGCTAG CTGCAGCGAC TACCGCATCA TCACAGCCTG TTGAACTCTT 300
CTGAGCAAGA GAAGGGGAGG CGGGGTAAGG GAAGTAGGTG GAAGATTCAG CCAAGCTCAA 360
GG ATG GAA GTG CAG TTA GGG CTG GGA AGG GTC TAC CCT CGG CCG CCG 407
Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Ary Pro Pro
l 5 lO 15
TCC AAG ACC TAC CGA GGA GCT TTC CAG AAT CTG TTC CAG AGC GTG CGC 455
Ser Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg
GAA GTG ATC CAG AAC CCG GGC CCC AGG CAC CCA GAG GCC GCG AGC GCA 503
Glu Val Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala Ser Ala
GCA CCT CCC GGC GCC AGT TTG CTG CTG CTG CAG CAG CAG CAG CAG CAG 55l
Ala Pro Pro Gly Ala Ser Leu Leu Leu Leu Gln Gln Gln Gln Gln Gln
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23
CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAA GAG 599
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Glu
ACT AGC CCC AGG CAG CAG CAG CAG CAG CAG GGT GAG GAT GGT TCT CCC 647
Thr Ser Pro Arg Gln Gln Gln Gln Gln Gln Gly Glu Asp Gly Ser Pro
9o 95
CAA GCC CAT CGT AGA GGC CCC ACA GGC TAC CTG GTC CTG GAT GAG GAA 695
0 Gln Ala His Arg Arg Gly Pro Thr Gly Tyr Leu Val Leu Asp Glu Glu
100 105 110
CAG CAA CCT TCA CAG CCG CAG TCG GCC CTG GAG TGC CAC CCC GAG AGA 743
Gln Gln Pro Ser Gln Pro Gln Ser Ala Leu Glu Cys His Pro Glu Arg
115 120 125
GGT TGC GTC CCA GAG CCT GGA GCC GCC GTG GCC GCC AGC AAG GGG CTG 791
Gly Cys Val Pro Glu Pro Gly Ala Ala Val Ala Ala Ser Lys Gly Leu
130 135 140
CCG CAG CAG CTG CCA GCA CCT CCG GAC GAG GAT GAC TCA GCT GCC CCA 839
Pro Gln Gln Leu Pro Ala Pro Pro Asp Glu Asp Asp Ser Ala Ala Pro
145 150 155
25 TCC ACG TTG TCC CTG CTG GGC CCC ACT TTC CCC GGC TTA AGC AGC TGC 887
Ser Thr Leu Ser Leu l~eu Gly Pro Thr Phe Pro Gly Leu Ser Ser Cys
160 165 170 175
TCC GCT GAC CTT AAA GAC ATC CTG AGC GAG GCC AGC ACC ATG CAA CTC 935
Ser Ala Asp Leu Lys Asp Ile Leu Ser Glu Ala Ser Thr Met Gln Leu
180 185 190
CTT CAG CAA CAG CAG CAG GAA GCA GTA TCC GAA GGC AGC AGC AGC GGG 983
Leu Gln Gln Gln Gln Gln Glu Ala Val Ser Glu Gly Ser Ser Ser Gly
35 195 200 205
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24
AGA GCG AGG GAG GCC TCG GGG GCT CCC ACT TCC TCC AAG GAC AAT TAC 1031
Arg Ala Arg Glu Ala Ser Gly Ala Pro Thr Ser Ser Lys Asp Asn Tyr
210 215 220
TTA GGG GGC ACT TCG ACC ATT TCT GAC AAC GCC AAG GAG TTG TGT AAG 1079
Leu Gly Gly Thr Ser Thr Ile Ser Asp Asn Ala Lys Glu Leu Cys Lys
225 230 235
GCA GTG TCG GTG TCC ATG GGC CTG GGT GTG GAG GCG TTG GAG CAT CTG 1127
0 Ala Val Ser Val Ser Met Gly Leu Gly Val Glu Ala Leu Glu His Leu
240 245 250 255
AGT CCA GGG GAA CAG CTT CGG GGG GAT TGC ATG TAC GCC CCA CTT TTG 1175
Ser Pro Gly Glu Gln Leu Arg Gly Asp Cys Met Tyr Ala Pro Leu Leu
260 265 270
GGA GTT CCA CCC GCT GTG CGT CCC ACT CCT TGT GCC CCA TTG GCC GAA 1223
Gly Val Pro Pro Ala Val Arg Pro Thr Pro Cys Ala Pro Leu Ala Glu
275 280 285
TGC A~A GGT TCT CTG CTA GAC GAC AGC GCA GGC A~G AGC ACT GAA GAT 1271
Cys Lys Gly Ser Leu Leu Asp Asp Ser Ala Gly Lys Ser Thr Glu Asp
290 295 300
25 .ACT GCT GAG TAT TCC CCT TTC AAG GGA GGT TAC ACC A~A GGG CTA GAA 1319
Thr Ala Glu Tyr Ser Pro Phe Lys Gly Gly Tyr Thr Lys Gly Leu Glu
305 310 315
GGC GAG AGC CTA GGC TGC TCT GGC AGC GCT GCA GCA GGG AGC TCC GGG 1367
Gly Glu Ser Leu Gly Cys Ser Gly Ser Ala Ala Ala Gly Ser Ser Gly
320 325 330 335
ACA CTT GAA CTG CCG TCT ACC CTG TCT CTC TAC AAG TCC GGA GCA CTG 1415
Thr Leu Glu Leu Pro Ser Thr Leu Ser Leu Tyr Lys Ser Gly Ala Leu
340 345 350
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GAC GAG GCA GCT GCG TAC CAG AGT CGC GAC TAC TAC AAC TTT CCA CTG 1463
Asp Glu Ala Ala Ala Tyr Gln Ser Arg Asp Tyr Tyr Asn Phe Pro Leu
355 360 365
GCT CTG GCC GGA CCG CCG CCC CCT CCG CCG CCT CCC CAT CCC CAC GCT 1511
Ala Leu Ala Gly Pro Pro Pro Pro Pro Pro Pro Pro His Pro His Ala
370 375 380
CGC ATC AAG CTG GAG A~C CCG CTG GAC TAC GGC AGC GCC TGG GCG GCT 1559
0 Arg Ile Lys Leu Glu Asn Pro ~eu Asp Tyr Gly Ser Ala Trp Ala Ala
385 390 395
GCG GCG GCG CAG TGC CGC TAT GGG GAC CTG GCG AGC CTG CAT GGC GCG 1607
Ala Ala Ala Gln Cys Arg Tyr Gly Asp Leu Ala Ser Leu His Gly Ala
400 405 410 415
GGT GCA GCG GGA CCC GGT TCT GGG TCA CCC TCA GCC GCC GCT TCC TCA 1655
Gly Ala Ala Gly Pro Gly Ser Gly Ser Pro Ser Ala Ala Ala Ser Ser
420 425 430
TCC TGG CAC ACT CTC TTC ACA GCC GAA GAA GGC CAG TTG TAT GGA CCG 1703
Ser Trp His Thr Leu Phe Thr Ala Glu Glu Gly Gln Leu Tyr Gly Pro
435 440 445
TGT GGT GGT GGT GGG GGT GGT GGC GGC GGC GGC GGC GGC GGC GGC GGC 1751
Cys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
450 455 460
GGC GGC GGC GGC GGC GGC GGC GGC GGC GAG GCG GGA GCT GTA GCC CCC 1799
Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Ala Gly Ala Val Ala Pro
465 470 475
TAC GGC TAC ACT CGG CCC CCT CAG GGG CTG GCG GGC CAG GAA AGC GAC 1847
Tyr Gly Tyr Thr Arg Pro Pro Gln Gly ~eu Ala Gly Gln Glu Ser Asp
35 480 485 490 495
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26
TTC ACC GCA CCT GAT GTG TGG TAC CCT GGC GGC ATG GTG AGC AGA GTG 1895
Phe Thr Ala Pro Asp Val Trp Tyr Pro Gly Gly Met Val Ser Arg Val
500 505 510
5 CCC TAT CCC AGT CCC ACT TGT GTC A~A AGC GAA ATG GGC CCC TGG ATG 1943
Pro Tyr Pro Ser Pro Thr Cys Val Lys Ser Glu Met Gly Pro Trp Met
515 520 525
GAT AGC TAC TCC GGA CCT TAC GGG GAC ATG CGT TTG GAG ACT GCC AGG 1991
0 Asp Ser Tyr Ser Gly Pro Tyr Gly Asp Met Arg Leu Glu Thr Ala Arg
530 535 540
GAC CAT GTT TTG CCC ATT GAC TAT TAC TTT CCA CCC CAG AAG ACC TGC 2039
Asp His Val Leu Pro Ile Asp Tyr Tyr Phe Pro Pro Gln Lys Thr Cys
545 550 555
CTG ATC TGT GGA GAT GAA GCT TCT GGG TGT CAC TAT GGA GCT CTC ACA 2087
Leu Ile Cys Gly Asp Glu Ala Ser Gly Cys His Tyr Gly Ala Leu Thr
560 565 570 575
TGT GGA AGC TGC AAG GTC TTC TTC AAA AGA GCC GCT GAA GGG AAA CAG 2135
Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala Ala Glu Gly Lys Gln
580 585 590
AAG TAC CTG TGC GCC AGC AGA AAT GAT TGC ACT ATT GAT AAA TTC CGA 2183
Lys Tyr Leu Cys Ala Ser Arg Asn Asp Cys Thr Ile Asp Lys Phe Arg
595 600 605
AGG APA AAT TGT CCA TCT TGT CGT CTT CGG AAA TGT TAT GAA GCA GGG 2231
30 Arg Lys Asn Cys Pro Ser Cys Arg Leu Arg Lys Cys Tyr Glu Ala Gly
610 615 620
ATG ACT CTG GGA GCC CGG AAG CTG AAG AAA CTT GGT AAT CTG AAA CTA 2279
Met Thr Leu Gly Ala Arg Lys Leu Lys Lys Leu Gly Asn Leu Lys Leu
625 630 635
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27
CAG GAG GAA GGA GAG GCT TCC AGC ACC ACC AGC CCC ACT GAG GAG ACA 2327
Gln Glu Glu Gly Glu Ala Ser Ser Thr Thr Ser Pro Thr Glu Glu Thr
640 645 650 655
ACC CAG AAG CTG ACA GTG TCA CAC ATT GAA GGC TAT GAA TGT CAG CCC 2375
Thr Gln Lys Leu Thr Val Ser His Ile Glu Gly Tyr Glu Cys Gln Pro
660 665 670
ATC TTT CTG AAT GTC CTG GAA GCC ATT GAG CCA GGT GTA GTG TGT GCT 2423
0 Ile Phe Leu Asn Val Leu Glu Ala Ile Glu Pro Gly Val Val Cys Ala
675 680 685
GGA CAC GAC AAC AAC CAG CCC GAC TCC TTT GCA GCC TTG CTC TCT AGC 2471
Gly His Asp Asn Asn Gln Pro Asp Ser Phe Ala Ala Leu Leu Ser Ser
690 695 700
CTC AAT GAA CTG GGA GAG AGA CAG CTT GTA CAC GTG GTC AAG TGG GCC 2519
Leu Asn Glu Leu Gly Glu Arg Gln Leu Val His Val Val Lys Trp Ala
705 710 715
AAG GCC TTG CCT GGC TTC CGC AAC TTA CAC GTG GAC GAC CAG ATG GCT 2567
Lys Ala Leu Pro Gly Phe Arg Asn Leu His Val Asp Asp Gln Met Ala
720 725 730 735
GTC ATT CAG TAC TCC TGG ATG GGG CTC ATG GTG TTT GCC ATG GGC TGG 2615
Val Ile Gln Tyr Ser Trp Met Gly Leu Met Val Phe Ala Met Gly Trp
740 745 750
CGA TCC TTC ACC AAT GTC AAC TCC AGG ATG CTC TAC TTC GCC CCT GAT 2663
Arg Ser Phe Thr Asn Val Asn Ser Arg Met Leu Tyr Phe Ala Pro Asp
760 765
CTG GTT TTC AAT GAG TAC CGC ATG CAC AAG TCC CGG ATG TAC AGC CAG 2711
Leu Val Phe Asn Glu Tyr Arg Met His Lys Ser Arg Met Tyr Ser Gln
770 775 780
-
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TGT GTC CGA ATG AGG CAC CTC TCT CAA GAG TTT GGA TGG CTC CAA ATC 2759
Cys Val Arg Met Arg His Leu Ser Gln Glu Phe Gly Trp Leu Gln Ile
785 790 795
ACC CCC CAG GAA TTC CTG TGC ATG A~A GCA CTG CTA CTC TTC AGC ATT 2807
Thr Pro Gln Glu Phe Leu Cys Met Lys Ala Leu Leu Leu Phe Ser Ile
800 805 810 815
ATT CCA GTG GAT GGG CTG AAA AAT CAA A~A TTC TTT GAT GAA CTT CGA 2855
0 Ile Pro Val Asp Gly Leu Lys Asn Gln Lys Phe Phe Asp Glu Leu Arg
820 825 830
ATG AAC TAC ATC AAG GAA CTC GAT CGT ATC ATT GCA TGC A~A AGA AAA 2903
Met Asn Tyr Ile Lys Glu Leu Asp Arg Ile Ile Ala Cys Lys Arg Lys
835 840 845
AAT CCC ACA TCC TGC TCA AGA CGC TTC TAC CAG CTC ACC AAG CTC CTG 2951
Asn Pro Thr Ser Cys Ser Arg Arg Phe Tyr Gln Leu Thr Lys Leu Leu
850 855 860
GAC TCC GTG CAG CCT ATT GCG AGA GAG CTG CAT CAG TTC ACT TTT GAC 2999
Asp Ser Val Gln Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe Asp
865 870 875
CTG CTA ATC AAG TCA CAC ATG GTG AGC GTG GAC TTT CCG GAA ATG ATG 3047
Leu Leu Ile Lys Ser His Met Val Ser Val Asp Phe Pro Glu Met Met
880 885 890 895
GCA GAG ATC ATC TCT GTG ~AA GTG CCC AAG ATC CTT TCT GGG AAA GTC 3095
Ala Glu Ile Ile Ser Val Gln Val Pro Lys Ile Leu Ser Gly Lys Val
goo 905 910
AAG CCC ATC TAT TTC CAC ACC CAG TGAAGCATTG GA~ACCCTAT TTCCCCACCC 3149
Lys Pro Ile Tyr Phe His Thr Gln
35 915 920
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CAGCTCATGC CCCCTTTCAG ATGTCTTCTG C~l~lLATAA CTCTGCACTA CTCCTCTGCA 3209
GTGCCTTGGG GAATTTCCTC TATTGATGTA CAGTCTGTCA TGAACATGTT CCTGAATTCT 3269
ATTTGCTGGG ~'l"l"L'l"l"l"l"l"l' CTCTTTCTCT CCTTTCTTTT TCTTCTTCCC TCCCTATCTA 3329
ACCCTCCCAT GGCACCTTCA GACTTTGCTT CCCATTGTGG CTCCTATCTG ~l~llllGAAT 3389
GGTGTTGTAT GCCTTTAAAT CTGTGATGAT CCTCATATGG CCCAGTGTCA AGTTGTGCTT 3449
GTTTACAGCA CTACTCTGTG CCAGCCACAC AAACGTTTAC TTATCTTATG CCACGGGAAG 3509
TTTAGAGAGC TAAGATTATC TGGGGAAATC A~AACAAAAA ACAAGCAAAC AAA~U~AA~ 3569
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1082 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: dou~le
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(ix) FEATURE:
(A) NAME/KEY: exon
35 (B~ LOCATION: 602.. 770
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(D) OTHER INFOR~L~TION: /note= "SEGMENT 1 OF 3.1'
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
AAGCTTCCCT TAACATACTA ACCCTTTACT TTCCCTGTTG TGTCCCTGAA AGGCCTCCTG 60
S TGCCTTTGGC TGCAGGTCCC GAACGTCCAG GCCATCTGTG CTATCTGCTT CGCGGTACCT 120
CACCAACGCA ACGTGAGGGT GGAGGGCAGA ACCTTGGTCC TGGCCTCTCA GCTTTTGTGG 180
GTTTCAGCCA GACCCTAGGT GTTATTTTAG TGCAACTTTG GTGTTTAATT TGAGGATGTG 240
TGTGGACCAG AAGGAGGGAC CAAAACATGA TTCTTTTCCC CATGGTCAGA TGATTA~ATT 300
TGAAGTTCTA AAAAATGCAG TTTGGTCCAA AGCTGTGTCC AATTGGGAAG AGAGA~AAAT 360
GCCCTGGAAA CCCCTCCCAG GCCTGGGACC ATCCTTCCTT AACCACCAGC CACCTCACAG 420
GCCCGCGGAC TGCGGGCATC ACCTGGGCAG GCTGTGCTTA CTCACTACCC GGGAACCCTG 480
TGCCCTGGAG CTGTCCTTCC TCTCTTCA~A GTGCATTTTG TGCGTTTGCT GGAAGAACCG 540
ACTACAGGTT TGTTCAATTT CTTACAGTCT TGA~AGCGCC ACAAGCAGCA GCTGCTGAGC 600
CATGGCTGAA GGGGA~ATCA CCACCTTCAC AGCCCTGACC GAGAAGTTTA ATCTGCCTCC 660
AGGGAATTAC AAGAAGCCCA AACTCCTCTA CTGTAGCAAC GGGGGCCACT TCCTGAGGAT 720
CCTTCCGGAT GGCACAGTGG ATGGGACAAG GGACAGGAGC GACCAGCACA GTAAGCCCAT 780
CTCTATGGCA CCCCCCTTCC CTTTCTGACA TCTTCTGTAG TCAAGGTGGG AGGAAGGTGC 840
ACATTTAAGT ACAGGTACTT GCTTCTCCAA GGTTCTATTC AGGCATGACA CATTCAGAGG 900
TGGAGTCACA TAAATGCGTA A~ATGTCTGG GA~ATGAA~A TAGGGACTTG TGGGGGCCAC 960
CACTTACCCA AACGTGTCCT ATTTCAAGTT TTTTAAAGCA CTCTCTGCTG ACCCAACAGA 1020
CA 02239976 l998-03-l7
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ACGGGCTGCC GGTGCTCAAT TGCTGTATGT TTTCCCAGGT TTCTGTAACT AGTGA~AGAT 1080
CT 1082
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 427 base pairs
(B) TYPE: nucleic acid
lC) STRANDEDNESS: double
(D) TOPOLOGY: l;n~
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYP~~ CAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 186..289
(D) OTHER INFORMATION: /note= "SEGMENT 2 OF 3. UNKNOWN
NUMBER OF BP AFTER SEGMENT 1."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CAGCTTTCTT TGGAAGGCAA AGA~AAAGGG ACTGTATTTC TATGTTTTGA TTAATCTGAG 60
GCTCATCCTG AGGGCTCCGT GA~ATGAATG AGCAGAATTT TCCATGGCCA ACTGTCCTGG 120
CTGCCGGGTC CTATCGGCAA AAGCGTAGTG TTTATTTACT TTTGCTCGTG TTATTTTTAT 180
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TCCAGTTCAG CTGCAGCTCA GTGCGGAAAG CGTGGGGGAG GTGTATATAA AGAGTACCGA 240
GACTGGCCAG TACTTGGCCA TGGACACCGA CGGGCTTTTA TACGGCTCAG TAAGTATGAA 300
GCTGACATGC TTCCAGACGT TGGCCAAGGT TTGAGGTTTC CAGA~ATCTT GTTACATGGA 360
GTGAGGCA~A CTATA~AGCA ACAATTAGTC TCTGTTTGTT A~ CCA GAAGGATTCC 420
CACCCTC 427
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 664 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
25 (vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(ix) FEATURE:
(A) NAME/KEY: exon
30 (B) LOCATION: 304.. 498
(D) OTHER INFORMATION: /note= "SEGMENT 3 OF 3. UNKNOWN
NUMBER OF BP AFTER SEGMENT 2."
35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
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TGAGGACTCT TAGAAGTGCT CTTATCAGTA GCATCTTAAT TACTTTACA~ TGGATTTTAA 60
ATGGA~AGGA AGTTTACAAT AATAGCA~AT GCATATTGAC AGCTCTTTAG TGCCCGGTGC 120
TGTTCTAAGT CCTTATGACT ACCCTGTGAA ATAAGTTCCA CCATGACCCC AATTTTCCTG l80
AAAAGGAGAC TGAGGCATGG AGAGCTTTAG TATTTTGCCC AATGTCACAC AGCTAGTA~A 240
TGGGGACCCC CATGTGAAAC TACTCACTGA TTGTCCTACT CTCTTGTGGT TTTATCTTTT 300
TAGCAGACAC CAAATGAGGA ATGTTTGTTC CTGGA~AGGC TGGAGGAGAA CCATTACAAC 360
ACCTATATAT CCAAGAAGCA TGCAGAGAAG AATTGGTTTG TTGGCCTCAA GAAGAATGGG 420
AGCTGCA~AC GCGGTCCTCG GACTCACTAT GGCCAGAAAG CAATCTTGTT TCTCCCCCTG 480
CCAGTCTCTT CTGATTAAAG AGATCTGTTC TGGGTGTTGA CCACTCCAGA GAAGTTTCGA 540
GGGGTCCTCA CCTGGTTGAC CCAAAAATGT TCCCTTGACC ATTGGCTGCG CTAACCCCCA 600
GCCCACAGAG CCTGAATTTG TAAGCAACTT GCTTCTAAAT GCCCAGTTCA CTTCTTTGCA 660
GAGC 664
(2) INFORMATION FOR SEQ ID ~0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPO~OGY: linear
(ii) MoLEcu-rlE TYPE: cDNA
35 (iii) HYPOTHETICAL: NO
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(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: ~YNl~llC OLIGONUCLEOTIDE
(ix) FEATURE:
(A) NAME/KEY: misc_~eature
(B) LOCATION: l..27
(D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS
927-953 OF SEQ ID NO.: l."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
15 CTGCTGCTGT TGCTGAAGGA GTTGCAT 27
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: ~YN'l'~'llC OLIGONUCLEOTIDE
(ix) FEATURE:
(A) NAME/KEY: misc_~eature
CA 02239976 l998-03-l7
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(B) LOCATION: 1 .21
(D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS
916-936 OF SEQ ID NO.: 1."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GGAGTTGCAT GGTGCTGGCC TCAGCACCA 29
10 ~(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: l; n
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: ~YN'l'~'l'lC OLIGONUCLEOTIDE
(ix) FEATURE:
(A) NAME/KEY: misc_~eature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /note= "AMTISENSE TO POSITIONS
927-947 OF SEQ ID NO.: 1.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
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~lGll~CTGA AGGAGTTGCA TAACTCCTT 29
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: ~YN'l'~'l'lC OLIGONUCLEOTIDE
(ix) FEATURE:
(A) NAME/KEY: misc_~eature
(B) LOCATION: l..25
(D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS
611-635 OF SEQ ID NO.: 2."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GGGCTGTGAA GGTGGTGATT TCCCC 25
