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
S 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 ~nti~en~e oligonucleotides and their use in human hyperplasia
and cancer therapy.
Background of the Invention
Trç~tment 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
trç~tn~ent~ introduced by Charles Huggins in the 1 940s. A rem~rk~kle relief of symptoms and
objective regression of bony met~t~es occurs under this endocrine therapeutic program.
Unfortunately, after a "golden period" which lasts roughly 18 months, regrowvth 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 collv~lllional regimen of combined chemotherapeutic
agents also is typically ineffective after the golden period, and a downhill clinical course follows,
t~ 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 ofthe disease, which is all too often (see, e.g., Gittes (1991);
and Catalona (1994)), the prospect of beneflt from available chemotherapy is gloomy.
Less severe but more comrnon than prostatic cancer is benign prostatic hyperplasia
(BPH). This condition may be a precursor to full blown prostatic cancer or may continue for
decades without evolving into the deadly carcinoma. Depending upon the degree of hypertrophy
and the age of the patient, trç~tment may range from "watchful waiting" to more aggressive
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approaches employing anti-androgen horrnonal therapy, tran~ resection, or radical
prostatectomy (see, e.g., Catalona (1994)).
Prostatic specific antigen (PSA) was first described by Wang et al. (1979) as a specific
marker for prostate tissue and was subsequently identified by Papsidero et al. (1980) as being
S present in the sera of prostate cancer patients. Since then, PSA in the sera has become the most
prevalent diagnostic marker for cancer of the prostate (see, e.g., Gittes (1991); Catalona (1994);
Oesterling (1995); and Pienta (1995)). Levels of serum PSA are also elevated in BPH but less so
than in progressed prostatic cancer. The complete arnino acid sequence of PSA was disclosed by
Watt et al. (1986) and the complete gene encoding PSA was disclosed by Lundwall (1989) and
10 Klobeck et al. (1989). PSA is a glycoprotein having a single polypeptide chain and a molecular
mass of appro~im~f~ly 34kDa. PSA is produced exclusively by epithelial cells of the prostate
and is localized to the rough endoplasmic reticulum and associated vesicles until it is secreted
into the acini and ducts ofthe prostate (see, e.g., Sinha, et al. (1987)). There PSA functions as a
neutral serine protease which serves to liquefy the semin~l coagulum by degrading seminal
15 vesicle proteins including fibronectin and semenogelin (see, e.g., Lilja (1985); Warhol and
Logtine (1985)). Higher serum PSA levels are correlated with the presence and progression of
prostate cancer. Despite variance between patients, PSA levels are useful both in monitoring the
progress of individual patients and as an indicator for diagnosing or staging prostate cancer (see,
e.g., El-Shirbiny (1994)).
Probasin is a prostate specific basic protein first isolated by Matuo et al. (1982) from rat
dorsolateral prostate. A cDNA to the rat probasin was disclosed by Spence et al. (1989) and
revealed two in-frame translation initiation codons which are believed to account for the secreted
and nuclear forms of the protein. The physiological role of probasin is unknown but it is a
heparin binding protein that co-purifies with heparin binding growth factor-l (HBGF-l) and is
25 positively regulated by androgen. Probasin appears to have minor mitogenic activity (0.2-1% of
HBGF- 1) but this may be an artifact of its co-purification with HBGF- 1 (Matuo et al. (1989)).
Summaly of the Invention
The present invention provides methods for keating a patient diagnosed as having benign
30 prostatic hyperplasia or a prostatic cancer. The methods include ~flmini~t~ring to the patient a
therapeutically effective amount of a composition comprising an ~nti~n~e oligonucleotide which
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selectively hybridizes to a PSA or probasin gene or mRNA sequence of the patient, thereby
inhibiting the expression of the PSA or probasin gene or mRNA sequence. This inhibition of the
PSA or probasin genes or mRNAs by ~nti~en~e 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 PSA ~nti~en~e oligonucleotides may comprise at least 10 consecutive bases from
SEQ ID NO.: 1, at least 10 consecutive bases from the joined exons of SEQ ID NO.: l; 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 sequences.
The probasin ~nti~çn~e oligonucleotides may comprise at least 10 consecutive bases from
SEQ ID NO.: 2, at least 10 consecutive bases from a genomic sequence corresponding to SEQ ID
NO.: 2, or oligonucleotides that hybridize to the complement~ of these sequences under
physiological conditions. More preferably, the ~nti~çn~e 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.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, and SEQ ID
NO.: 8.
In preferred embo-liment~, all of the above-described oligonucleotides are modified
oligonucleotides. In one set of embodiments, the modified oligonucleotide includes at least one
synthetic intermlcleoside linkage such as a phosphorothioate, alkylphosphonate,
phosphorodithioate, phosphate ester, alkylphosphonothioate, phosphor~micl~tç~ carbamate,
carbonate, phosphate triester, ~çet~midate, or carboxymethyl ester.
In other embo~liment~ with modified oligonucleotides, the modified oligonucleotide has
at least one low molecular weight organic group covalently bound to a phosphate group of said
oligonucleotide. In another set of embo-liment~, 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.,
aminoethyl, aminopropyl, aminohydroxyethyl, aminohydroxypropyl), small saccharides or
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glycosyl groups.
In another set of embo-1iment~ the modified oligonucleotide has covalently ~tt~.he~l
thereto a prostate-targeting compound such as an androgen, androgen derivative, estrogen,
estrogen derivative, estramustine, emcyt or estracyt.
In preferred embo~lim~ntc, the antisense oligonucleotides are ~flminict~red intravenously
at a dosage between 1.0 ,ug and 100 mg per kg body weight of the patient.
The present invention also provides for any or all of the above-described ~nti~n~e
oligonucleotides, including the various modified oligonucleotides, in a ph~rm~ceutical
composition. The antisense oligonucleotides are admixed with a sterile ph~rm~elltically
10 acceptable carrier in a therapeutically effective amount such that the isolated ~nti~e~e
oligonucleotide selectively hybridizes to the PSA or probasin gene or mRNA sequence when
~1mini~tered to a patient. A phz~rmz~eeutical kit is also provided in which such a pharmaceutical
composition is combined with a ph~rm~r~eutically acceptable carrier for intravenous
~lmini~tration.
Detailed D~sL ;l.lion of the Invention
The present invention provides new methods for tbe tre~tment of cancer of the prostate
and ph~rm~t~elltical compositions useful therefor. It is now disclosed that ~nti~n~e
oligonucleotides compl~ment~ry to genes which are expressed only or predomin~ntly in prostatic
20 cells are effective for inhibiting the growth of and/or killing hyperplastic or cancerous cells of
prostatic origin. In particular, the present invention provides oligonucleotides, including
modified oligonucleotides, which have ~nti~n~e homology to a sufficient portion of either the
PSA or probasin gene such that they inhibit the expression of that gene. The ex~ s~ion of both
of these genes is believed to be tissue specific to the prostate. Surprisingly, inhibition of either
25 of these genes, both of which encode secreted proteins with no known function within prostate
cells, inhibits the growth of these cells. Because the ~nti~en~e oligonucleotides of the invention
can be ~flmini.~tered systemically but selectively inhibit prostate cells, the present invention has
particular utility in late stage prostate cancer which has met~t~i7~d
Definitions
In order to describe more clearly and concisely the subject matter of the present
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invention, the following definitions are provided for specific terms used in the claims appended
hereto:
~ . As used herein, the abbreviation "PSA" refers to the prostatic specific antigen well
known in the art and described in the various references cited herein. Genomic DNA sequences
5 of the human PSA gene were disclosed in Lundwall (1989) and Klobeck et al. (1989). The
Klobeck et al. (1989) sequence is available on GenBank (Accession number X14810) and is
reproduced here as SEQ. ID NO.: 1. The translation initiation codon of this gene is found at base
positions 401-403 and the stop codon is at positions 5566-5568 of SEQ ID NO.: 1. The gene
consists of five exons which are indicated on SEQ. ID NO.: 1. A TATA signal is found at
10 positions 332-338 and a transcriptional start region appears at positions 355-365. As will be
obvious to one of ~ rdi~ skill in the art, other alleles of the PSA gene, including other human
alleles and homologues from other m~mm~ n species, encoding a PSA protein and hybridizing
to SEQ ID NO.: 1 under stringent hybridization conditions, will exist in natural populations and
are embraced by the term "PSA gene" as used herein. A slightly different sequence for PSA is
15 also available on GenBank (Acession number M27274). The PSA gene of the invention is
intt~ntlf~l to encompass all such sequences.
Probasin. As used herein, the term "probasin" refers to the probasin protein known in the
art and described in the various references cited herein. A cDNA to one allele of the rat probasin
gene was disclosed in Spence, et al. (1989). The Spence et al. (1989) sequence is available on
20 GçnR~nk (Accession nurnber M27156) and is reproduced here as SEQ. ID NO.: 2. This gene
has two potential translation initiation start codons which are in frame with each other. The first
is at positions 41-43 of SEQ ID NO. 2 and the second is at positions 92-94. The stop codon is at
positions 572-574. The bases between the first and second initiation codons encode a
hydrophobic sequence con~i~çnt with a secretory signal sequence. Thus, it is believed that the
25 initiation of translation from the first start codon leads to production of the secreted form of
probasin whereas translation from the second results in the nuclear form of the protein. As used
herein, the term "probasin gene" is specifically int~n~ to include a gene encoding either or
both forms of the probasin protein. In addition, as will be obvious to one of ordinary skill in the
art, other alleles of the probasin gene, including other human alleles and homologues from other
30 m~nnm~ n species, encoding a probasin protein and hybridizing to SEQ ID NO.: 2 under
stringent hybridization conditions, will exist in natural populations and are embraced by the term
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"probasin gene" as used herein.
Anti~ence Oli~onucleotides. As used herein, the term "~nti~en~e oligonucleotide" or
"zlnti~en~e" describes an oligonucleotide that is an oligoribonucleotide,
5 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 a "PSA-~nti~Pn~e oligonucleotide" and by a
"probasin-antisense oligonucleotide" are meant oligonucleotides which hybridize under
10 physiological conditions to the PSA gene/mRNA or probasin gene/mRNA and, thereby, inhibit
transcription/translation of the PSA and probasin genes/mRNAs, respectively. The antisense
molecules are de~i~ned so as to interfere with transcription or translation of PSA or probasin
upon hybridization with the target. Those skilled in the art will recognize that the exact length of
the antisense oligonucleotide and its degree of complement~rity will depend upon the specific
15 target selected, including the sequence of the target and the particular bases which comprise that
sequence. It is pler~ d that the ~nti~n~e 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~ent hybri-li7~tion conditions. As used herein, the term "stringent hybridization
20 conditions" means hybridization conditions from 30~C-60~C and from Sx 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
25 sequence and not to s~lkst~nti~lly dirr~ 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 hybri~1i7~tion is first observed, one of ol.l~ ,~y skill in the art can, without undue
30 experiment~tion, ~letermine conditions which will allow a given sequence to hybridize only with
identical sequences. Suitable ranges of such stringency conditions are described in Krause,
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M.H.. and S.A. Aaronson, Methods in En7ymolo~y~ 200:546-556 (1991). As used herein with
respect to in-~ivo hybridi_ation conditions, the term "physiological conditions" is considered
functionally equivalent to the in vitro stringent hybridization conditions.
I. nesi~n of PSA z~nd Probasin Antisense Oli~onucleotides
The present invention depends, in part, upon the discovery that the selective inhibition of
the expression of PSA or probasin by slnti~en~e oligonucleotides in prostatic cells effectively
inhibits cell growth and/or causes cell death.
Based upon SEQ ID NO.: 1 and SEQ ID NO.: 2, or upon allelic or homologous genomic
or cDNA sequences, one of skill in the art can easily choose and synthe~i7t? any of a number of
a~p~ iate ~nti~en~e molecules for use in accordance with the present invention. In order to be
sufficiently selective and potent for PSA or probasin inhibition, such ~nti~çn~e oligonucleotides
should comprise at least 10 and, more preferably, at least 15 consecutive bases which are
complement~ry to the PSA or probasin mRNA transcripts. Most preferably, the ~nti~n~e
oligonucleotides comprise a complement~ry sequence of 20-30 bases. Although
oligonucleotides may be chosen which are ~nti~n~e to any region of the PSA or probasin genes
or mRNA transcripts, in ~l.,r~ ,d embofliment~ the ~nti~çn~e oligonucleotides correspond to N-
termin~l or 5' U~:~LIc~LIll sites such as translation initiation, transcription initiation or promoter
sites. In addition, 3'-untr~n~l~tto~l regions or telomerase sites may be targeted. Targeting to
mRNA splicing sites has also been used in the art but may be less ~l~re~lled if ~It~rn~tive mRNA
splicing occurs. In addition, the PSA or probasin ~nti~çn~e 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. With respect to probasin, an N-tçrmin~l antisense
oligonucleotide may be targeted to either the first or the second initiation codon so as to interfere
with translation of both forms or just the secreted form of probasin. Finally, although, SEQ ID
NO.: 1 discloses a genomic DNA sequence and SEQ ID NO.: 2 discloses a cDNA sequence, one
of ordinary skill in the art may easily derive the cDNA corresponding to the joined exons of SEQ
ID NO.: 1 and may easily obtain the genomic DNA sequence corresponding to SEQ ID NO.: 2.
Thus, the present invention also provides for ~nti~n~e oligonucleotides which are
complement~ry to the cDNA corresponding to SEQ ID NO.: 1 and the genomic DNA
corresponding to SEQ ID NO.: 2. Similarly, slnti~.on~e to allelic or homologous cDNAs and
genomic DNAs are enabled without undue expçriment~tion.
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As will be understood by one of ordinary skill in the art, the ~nfi~en~e oligonucleotides of
the present invention need not be perfectly complement~ry to the PSA or probasin genes or
mRNA transcripts in order to be effective. Rather, some degree of mi~m~t~hes will be
acceptable if the ~nti~en~e oligonucleotide is of sufficient length. In all cases, however, the
oligonucleotides should have sufficient length and compl~ment~rity so as to hybridize to a PSA
or probasin tr~n~eriI-t under physiological conditions. Preferably, of course, mi~m~tc hes are
absent or minim~l In addition, although it is not l cco" . " ,en~ l, the ~nti ~n~e oligonucleotides
rnay have one or more non-complement~ry sequences of bases inserted into an otherwise
complement~ry ~nti~en~e oligonucleotide sequence. Such non-complementary sequences may
10 "loop" out of a duplex formed by a PSA or probasin transcript and the bases fl~nkinp; the non-
complement~ry region. Therefore, the entire oligonucleotide may retain an inhibitory effect
despite an a~palcnlly low percentage of complement~rity. 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
15 over and forming a duplex with itself. The duplex region, which is preferably at least 4-6 bases
joined by a loop of 3-6 bases, stabilizes the oligonucleotide against degradation. These self-
stabilized oligonucleotides are easily de~ignf~d by adding the inverted complement of a 5' or 3'
PSA or probasin sequence to the end of the oligonucleotide (see, e.g., Table 1, SEQ ID NO.: 5;
Tang, J.-Y., et al. (1993) Nuçleic Acids Res. 21:2729-2735).
In one set of embo~1iment~, the PSA and probasin ~nti~.on~e oligonucleotides of the
invention 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 intern~lcleoside linkage.
These oligonucleotides may be prepared by art recognized methods which may be carried out
25 m~nn~lly or by an automated synthesi7er.
In ~ r~ d embo-liment~, however, the ~nticen~e 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 hybricli7ing to their target but which enhance
their stability or targeting to prostatic cells or which otherwise enhance their therapeutic
30 errecLiv~;ness. The term "modified oligonucleotide" as used herein describes an oligonucleotide
in which ( 1 ) at least two of its nucleotides are covalently linked via a synthetic internucleoside
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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~rnnleleoside linkages are phosphorothioates, alkylphosphonates,
5 phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, car~m:~tee,
carbonates, phosphate triesters, ~cet~mil1~te, 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 or aliphatic groups (e.g.,
10 aminoethyl, aminopropyl, aminohydroxyethyl, aminohydro~y~lopyl), a small saccharides or
glycosyl groups. Other low molecular weight organic modifications include additions to the
int~rn~lcleoside phosphate linkages such as cholesteryl or fii~mine compounds with varying
numbers of carbon residues between the amino groups and te~nin~l ribose. Oligonucleotides
with these linkages or other modifications can be prepared according to known methods (see,
15 e.g., Agrawal and Goodchild (1987); Agrawal et al. (1988); Uhlmann et al. (1990); Agrawal et
al. (1992); Agrawal (1993); and U.S. Pat. No. 5,149,798).
The term "modified oligonucleotide" also encomr~eees oligonucleotides with a
covalently modified base andlor sugar. For example, modified oligonucleotides include
oligonucleotides having backbone sugars which are covalently attached to low molecular weight
20 organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at
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. Altern~tively, the modified oligonucleotides may be branched
oligonucleotides. Unoxidized or partially oxidized oligonucleotides having a substitution in one
25 or more nonbridging oxygen per nucleotide in the molecule are also considered to be modified
oligonucleotides.
Also considered as modified oligonucleotides are oligonucleotides having prostate-
hlg, nuclease resistance-conferring, or other bulky substituents and/or various other
- structural modifications not found in vivo without human intervention. The androgen receptor
30 and other hormonal receptor sites on prostate cells allow for targeting slntieenee oligonucleotides
specifically or particularly to prostatic cells. Att~rhment of the ~ntieen~e oligonucleotides by a
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molecular "tether" (e.g., an alkyl chain) to e~7~ , 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 (Forsgren7 et al.
(1979)). Similarly, one may covalently attach androgen7 estrogen, androgen or estrogen
S derivatives7 or other prostate cell ligands to ~nti~çn~e oligonucleotides using tethers and
conjugating linkages for prostatic targeting. Finally7 one may of course covalently attach other
chemotherapeutic agents (e.g., dexamethasone7 vinblastine7 etoposide) to the ~nti~n~e
oligonucleotides for enh~nrerl effect.
The most plc~rt;ll~,d modified oligonucleotides are hybrid or chimeric oligonucleotides in
10 which some but not all of the phosphodiester linkages7 bases or sugars have been modified.
Hybrid modified ~nti.~en~e oligonucleotides may be composed7 for example, of stretches of ten
2'-O-alkyl nucleotides or ten phosphorothioate synthetic linkages at the 5' and/or 3' ends, and a
segment of seven unmodified oligodeoxynucleotides in the center, or of similar te7 rnin~l
segments of alkyl phosphonates, with central P=S or P=O oligonucleotides (Agrawal7 et al.
15 (1990); Metelev7 et al. (1994)). The ~;ullell~ly most~l~r~ d modified oligonucleotides are 2'-O-
methylated hybrid oligonucleotides. Since degradation occurs mainly at the 3' end7 secondarily
at the 5' end, and less in the middle, unmodified oligonucleotides located at this position can
activate RNase H, and yet are degraded slowly. Furthermore, the Tm of such a 27-mer is
approximately 20 ~C higher than that of a 27-mer all phosphorothioate oligodeoxynucleotide.
20 This greater affinity for the targeted genomic area can result in greater inhibiting efficacy.
Obviously7 the number of synthetic linkages at the termini need not be ten and synthetic linkages
may be combined with other modifications7 such as alkylation of a 5' or 3' phosphate7 or 2'-O-
alkylation. Thus, merely as another example7 one may produce a modified oligonucleotide with
the following structure7 where B L~reSell~ any base7 R is an alkyl7 aliphatic or other substituent7
25 the subscript S l~piesell~ a synthetic (e.g. phosphorothioate) linkage7 and each n is an
independently chosen integer from 1 to about 20:
OH
5 (BS)nBBBB- ... -BBBB(BS)nB--P=03'
O--R
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II. Producte and Methods of Treatment for P~PH 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
invention are especially useful for late stage prostate cancer in which metastases have occurred
5 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 substitute for more radical procedures such as transurethral resection, radical
prostatectomy, or physical or chemical castration. The products of the present invention include
the isolated ~ntie~nee oligonucleotides described above. As used herein, the term "isolated" as
10 applied to an ~ntie~nee oligonucleotide means not covalently bound to and physically separated
from the 5' and 3' sequences which flank the corresponding ~ntie~nee sequence in nature.
~ lminietration of the PSA or probasin ~ntieçnee oligonucleotides may be oral,
intravenous, pa~ ldl, cutaneous or subcutaneous. For BPH or when the site of a prostatic
tumor is known, the ar1minietration also may be localized to the prostate or to the region of the
15 tumor by injection to or perfusion of the site.
PSA or probasin ~ntiePnee oligonucleotides may be ~lminietered as part of a
rh~rm~reutical composition. Such a ph~rm~-~e~ltical composition may include the ~ntie~nee
oligonucleotides in combination with any standard physiologically and/or phslrm~- eutically
acceptable carriers which are known in the art. The compositions should be sterile and contain a
20 thcld~t;ulically effective amount of the ~ntieenee oligonucleotides in a unit of weight or volume
suitable for ~tlminietration to a patient. The term "ph~ e~ltically acceptable" means a non-
toxic material that does not h.L~.r~.e with the effectiveness of the biological activity of the active
ingredients. The term "physiologically acceptable" refers to a non-toxic material that is
compatible with a biological system such as a cell, cell culture, tissue, or orp~niem The
25 characteristics of the carrier will depend on the route of ~tlminietration. Physiologically and
ph~ eutically acceptable carriers include ~lihlente, fillers, salts, buffers, stabilizers,
solubilizers, and other m~t~ri~le which are well known in the art. The I~hs~nnsl~eutical
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
30 included in the ph~rm~t~eutical composition to produce a synergistic effect or to minimi7~- side-
effects caused.
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The ph~rm~eelltical composition of the invention may be in the form of a liposome in
which the PSA or probasin antieçnee oligonucleotides are combined, in addition to other
ph~rrn~eeutically acceptable carriers, with amphipathic agents such as lipids which exist in
aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers which are
S in aqueous solution. Suitable lipids for liposomal fo~n~ ti-~n include, without limitation,
monoglycerides, diglycerides, s--lf~ti~lce lysolecithin, phospholipids, saponin, bile acids, and the
like. Ple~dl~LIion 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~rm~eeutical composition of the invention may further include compounds such as
cyclo-lçxtrine and the like which enh~nee delivery of oligonucleotides into cells. When the
composition is not ~-lnninietered systemically but, rather, is injected at the site of the target cells,
cationic detergents (e.g. Lipofectin) may be added to enh~n~e uptake.
When a th~ uLically effective amount of PSA or probasin ~ntieçnee oligonucleotides is
~iminietered orally, the oligonucleotides will be in the form of a tablet, capsule, powder, solution
or elixir. When ~riminietered in tablet form, the ph~rm~eeutical composition of the invention
may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and
powder may contain from about 5 to 95% of the PSA and/or probasin ~ntiet?nee oligonucleotides
and preferably from about 25 to 90% of the oligonucleotides. When ~rlminietered in liquid form,
20 a liquid carrier such as water, petroleum, 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
pharmaceutical composition may further contain physiological saline solution, dextrose or other
saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
When ~t1minietered in liquid form, the ph~rm~eelltical composition may contain from about 0.5
25 to 90% by weight of a PSA and/or probasin ~ntieenee oligonucleotide and preferably from about
1 to 50% of the oligonucleotide.
When a tht;l~ulically effective amount of a PSA or probasin ~ntieçnee oligonucleotide
is ~1minietered by intravenous, cutaneous or subcutaneous injection, the oligonucleotides will be
in the form of a pyrogen-free, ~ dlly acceptable aqueous solution. The ~l~p~u~lion of such
p~ lly acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is
within the skill in the art. A ~ rt;ll~d ph~rm~celltical composition for intravenous, cutaneous, or
CA 02232390 1998-03-17
W O 97/11172 13 PCT~US96/15123
subcutaneous injection should contain, in addition to the ~ntis~n~e oligonucleotides, an isotonic
vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and
Sodiurn Chloride Injection, Lactated Ringer's Injection, or another vehicle as known in the art.
The phzlrm~eeutical composition of the present invention may also contain stabilizers,
S preservatives, buffers, antioxitl~nt~, or other additives known to those of skill in the art.
In ~lcr~.led embo~1iments, when the target cells are readily ~ces~ihle, ~-iminictration of
the ~nti~ense oligonucleotides is localized to the region of the targeted cells in order to m~imi7.o
the delivery of the ~nti~ense and to minimi7e the amount of ~nti~en~e needed per trç~tment
Thus, in one ~lcr~llcd embodiment, ~-lministration is by direct injection at or perfusion of the
10 site of the targeted cells, such as a tumor. ~lt~rnsltively, the antisense oligonucleotides may be
adhered to small particles (e.g., microscopic gold beads) which are impelled through the
membranes of the target cells (see, e.g., U.S. Pat. No. 5,149,655).
In another series of embo-liments, a recombinant gene is constructed which encodes a
PSA or probasin antisense oligonucleotide and this gene is introduced within the targeted cells
15 on a vector. Such a PSA or probasin ~ntisPnse gene may, for example, consist of the normal
PSA or probasin sequence, or a subset of the normal sequences, operably joined in reverse
orientation to a promoter region. An operable ~ntis~n~e gene may be introduced on an
intç~r~tion vector or may be introduced on an expression vector. In order to be most effective, it
is ~rcre~l~d that the ~ntisçnse sequences be operably joined to a strong eukaryotic promoter
20 which is inducible or constitutively expressed.
In all of the above-described methods of tre~tment~ the PSA and/or probasin :~nti~ense
oligonucleotides are ~t1mini.stered in therapeutically effective amounts. As used herein, the term
"thc~d~culically effective amount" means that amount of antisense which, under the conditions of
~-iministration, including mode of ~1ministration and presence of other active components, is
25 sufficient to result in a me~ningful patient benefit, i.e., the killing or inhibition of the growth of
target cells.
The amount of PSA andlor probasin ~nti~Pnse oligonucleotides in the pharrnaceutical
composition of the present invention will depend not only upon the potency of the antisense but
also upon the nature and severity of the condition being treated, and on the nature of prior
treatments which the patient has undergone. Ultimately, the attending physician will decide the
amount of ~nti~en~ç with which to treat each individual patient. Initially, the ~tten~lin~ physician
CA 02232390 1998-03-17
W O 97/11172 14 PCTAJS96/15123
will ~Amini~ter low doses of the inhibitor and observe the patient's response. Larger doses of
antisense may be a~lmini~tered until the optimal therapeutic effect is obtained for the patient, and
at that point the dosage is not increased further. In preferred embo~iment~, it is contemplated
that the various ph~rm~eutical compositions used to practice the method of the present
5 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~rrn~eutical compositions of the present
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
10 adverse side effects (e.g., rapid lowering of blood pressure), slow intravenous ~lmini~tration is
p.cr~ d. Thus, i~ venous ~1mini~tration of th~ ulically effective amounts over a 12-24
hour period are contemplated. Ultimately the att~n~lin~ physician will decide on the ~ o~liate
duration of intravenous therapy using the ph~rm~eutical composition of the present invention.
The following examples of the use of PSA and probasin ~nti~çn~e are presented merely to
15 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 construed as limitin~ of the invention but, rather, as indicative of the wide range of
oligonucleotides which may be employed. As will be obvious to one of oLdil-a y skill in the art
in light of the present disclosure, a great many equivalents to the presently disclosed ~nti~en~e
oligonucleotides and disclosed methods are now available. In particular, other ~nti~en~e
oligonucleotides substantially complçment~ry to subsets of SEQ ID NO.: 1 or SEQ ID NO.: 2,
and chemical modifications of the same which do not prevent hybridization under physiological
conditions, are contemplated as equivalents of the examples presented below. In general, the use
of prostate specific ~nti~n~e oligonucleotides is contemplated as a method of selectively
inhibiting the growth of or killing prostatic cells.
Experimental Examples
Three permanent cell lines of human prostatic cancer were grown in monolayer culture:
LNCaP, PC3-1435, and DU145, all obtained from the American Type Culture Collection. The
30 LNCaP cells grow as stellate cells in a monolayer, retain hormone sensitivity and, of particular
i..lpol~lce, secrete PSA into the tissue culture medium (Oesterling (1995)). Cells were grown
CA 02232390 1998-03-17
W O 97/11172 PCTAJS96/15123
in Dulbecco's medium supplemented with 10 percent fetal calf serum, glllt~m~tç7 pyruvate,
penicillin and streptomycin, in 25-150 cm flasks, incubated at 37~C in 6 percent CO2-air.
A number of PSA and probasin ~ntieçnee oligonucleotides were tested for their inhibitory
effect on prostatic cells. The base sequences of these oligonucleotides are disclosed as SEQ ID
5 NO.: 3 through SEQ ID NO.: 9. SEQ ID NO.: 3 is antisense to positions 92-118 of the probasin
gene (SEQ ID NO.: 1). SEQ ID NO.: 4 is antisense to a region upstream of the probasin gene at
positions 76-99. SEQ ID NO.: 5 is a self-stabilized or hairpin oligonucleotide. The first 21
bases are complement~ry to positions 80-100 ofthe probasin gene. The rem~inin~ eight are
identical to positions 84-91 of the gene, allowing formation of a 3' hairpin. SEQ ID NO.: 6 is
10 another self-stabilized ~ntie~nee oligonucleotide. The first 21 bases ofthis oligonucleotide are
complement~ry to positions 92- 112 of the probasin gene. The rem~ining eight are identical to
positions 96-103 of the gene, allowing for formation of a 3' hairpin. SEQ ID NO.: 7 and SEQ ID
NO.: 8 are antisense sequences corresponding to positions 401-427 and 384-410 ofthe PSA
gene.
Table 1 shows some of the zmtiet-nee oligonucleotides tested. The numbers at the left of
each sequence correspond to the sequence numbers in the sequence listing. ~ntieenee
oligonucleotides with unmodified or natural intPrmlcleoside linkages (P=O) and oligonucleotides
with all phosphorothioate synthetic linkages (P=S) were tested. In addition, modified
oligonucleotides were tested in which just the ttormin~l two phosphodiester linkages at each end
20 had been replaced by phosphorothioate synthetic linkages (shown as a subscript S between
nucleotides in Table 1) and/or in which small aliphatic chemical groups (e.g., 2-hydroxy-3-
amino-propyl) were added to the 3' termin~l phosphate.
Growth of the PC3-1435 cell line in tissue culture monolayers was conei ctçntly inhibited
by addition of phosphorothioate-modified oligodeoxynucleotides targeted against the PSA or
25 probasin genes and incubation for 24-48 hours thereafter. As the concentration of modified
oligonucleotides is decreased from the 10-20 ~M level, most effective inhibition occurs with
specific zlntieçnee oligodeoxynucleotides at the 2-5 IlM level, as contrasted with miem~trhed
oligodeoxynucleotides (see Tables 2 and 3).
While the effects on cell growth (i.e. cell numbers) are readily manifest, visual substage
30 microscopy of wells revealed additional features of the inhibition events using PSA antisense
oligonucleotides against PC3-1435 cells. The first evidence of ~ntie~nee inhibition is rupture of
CA 02232390 1998-03-17
W O 97/11172 PCTAUS96/15123
16
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 ex~min~tl 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
5 DNA, in other words more drastic than the i, . ,p~ i . " ,ent of DNA synthesis.
PSA protein was measured in the tissue culture me~ m in which LNCaP cells were
grown in multi-well plates. This provided a qll~ntit~tive assay, using a '25I-anti-PSA labeled
antibody sandwich assay. We measured PSA levels in tissue cultures in as little as 2.5 ,ul of
incubation medium by means of the iodinated antibody ~ technique. The labeled antibody is
10 available from Hybritech (San Diego, CA). Table 5 shows the results from two negative controls
(no tre~tment), two positive controls employing an albiL~u y oligonucleotide which is ~nti~ence
to a portion of the HIV genome, and the SEQ ID NO.: 8 modified oligonucleotide of Table 1.
Using this sandwich assay, the PSA output into the tissue culture meflillm of LNCaP cells was
reduced 51 percent at the 5 ,uM level, and 45 percent at the 2.5 ~M level at a 48 hour time period
15 when the PSA gene was targeted by ~nti~n~e oligonucleotides.
Each of the above-mentioned references and patents is hereby incorporated by reference.
TABLE 1
~ QC Oligonucleotides
Sequence Target
#3 5'CTT-TTT-GAG-ATT-CTT-GTC-TGT-CAT-CAT3' Probasin, P=S
#3 5'CTT-TTT-GAG-ATT-CTT-GTC-TGT-CAT-CAT3' Proba~in, P=O
#4 5'GTC-ATC-ATA-CTG-GAG-ACA-CCT-AGC3' Probasin-
upstream, P=S
#5 5'TGT-CAT-CAT-ACT-GGA-GAC-ACC-TCT-CCA-GT3 Probasin 3'end
hairpin, P=S
#6 5GAG-ATT-CTT-GTC-TGT-CAT-CAT-TGA-CAG-AC3' Probasin 3~end
hairpin, P=S
30 #7 5GGT-GAG-GAA-GAC-AAC-CGG-GAC-CCA-CAT3' PSA, P-S
#8 5'GGA-CCC-ACA-TGG-TGA-CAC-AGC-TCT-CCG3' PSA, P=S
77418.1
CA 02232390 1998-03-17
W O 97/11172 17 PCT~US96/15123
TABLE 2
3H-thymidine incorporation into DNA PC3-1435
S human prostate cancer tissue culture
Gent-~ T~rgetedConcentration (,uM) CPM~ % inhibition
Control (no oligo) -- 38,000 0
Probasin (P = S) 20 13,700 64
1 8,000 52
Mi~m~t~,h (P = S) 20 20,000 47
27,000 30
15 t Averages of 3 separate wells
TABLE 3
Comparisons of degree of inhibition of DNA synthesis
in human PC3-1435 prostate cancer tissue cultures
20 for one target and a mix of targets
Genes t~r~etedConcentration (~lM) CPM ~ % ;nhibition
Control (no oligo) -- 14,700 0
Mi~m~tc,h 20 6,990 51
10,750 27
Mix* 20 4,930 66
6,054 59
30 * Mix: Probasin, PSA, PSA upstream, PSA farther U~Ll.,a~
S ~LM each at 20~1M total; 1.25,uM each at S ~M total.
~ Averages of 3 separate wells.
CA 02232390 1998-03-17
W O 97/11172 18 PCTAUS96/15123
TABLE 4
Morphological Comparison of Treated and Control Cells
Concentr~tion ~M
Gene Tar~et 20 10 5 2
PSA gene (P=S) 4+ 3+ 2-1/2+ 1+
~i~m~tc.h (P=S) 1-1/2+ 1/2+ 0 0
Observation 24 hours after oligonucleotide addition. Damage: 4+ dev~t~tin~; 3+ severe; 2+
serious, 1+ visible, 1/2+ slight, 0 none
TABLE ~
PSA Levels in Media of Cultured LNCaP Cells 24 hours after
Oligodeoxynucleotide Phosphorothioate Treatment
S~m~7leNo. Des.,liy~ion CPM PSA ~ng/mV
No tre~tment 11,940 33
2 No tre~tment 11,389 31
3 + Control 5 ~M 8,311 23
4 ~ Control 1 ~M 8,892 25
PSA antisense 5 ,uM 5,765 16
6 PSA ~nti~çn~e 1 IlM 5,375 17
CA 02232390 1998-03-17
W O 97/11172 PCTAUS96/15123
Ref~,. . ecs
Agrawal (ed.) Meth. Mol. Biol., Humana Press, Totowa, NJ (1993) Vol. 20.
Agrawal and Goodchild (1987) Tetrahedron T,ett. 28:3539-3542.
Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083.
5 Agrawal et al.(l990) Proc. Natl. Acad. Sci. (USA) 87: 1401-1405.
Agrawal et al. (1992) TrendsBiotechnol. 10:152-158.
Catalona(1994)N.E. J. Med. 331:996-1004.
El-Shirbiny (1994) Adv. Clin. Chem. 31:99-133.
Forsgren et al. (1979) Cancer Res. 39:5155-5164.
10 Gittes (1991) N F, J. Med. 324: 236-245.
Klobeck et al. (1989) Nucleic Acids Res. 17(10):3981.
Lilja (1985) J. Clin. Invest. 76:1899-1903.
Lundwall (1989) Biochem. Bio,~hys. Res. Commun. 161(3):1151-1159.
Matuo et al. (1982) Biochem. Biophys. Res. Comml-n 109:334-340.
15 Matuo et al. (1989) In Vitro Cell. Devel. Biol. 25(6):581-584.
Metelev et al. (1994) Bioor~. Medicinal Chem. T,ett. 4: 2929-2934.
Oesterlin~ (1995) Mich~n Onc. J. 6:8-12.
- Papsidero et al. (1980) Cancer Res. 40:2428-2432.
Pienta (1995) Michi~n Onc. J. 6:4-7.
20 Sainio et al. (1994) Cell Mol. Neurobiol. 14(5):439-457.
Sheridan and Tew (1991) ~ncer Surveys 11 :239-254.
Sinha et al. (1987) Cancer 60:1288-1293.
Spence et al. (1989) Proc. Natl. Acad. Sci. (USA) 86:7843-7847.
Uhlm~nn et al. (1990) Chem. Rev. 90:534-583.
25 Wang et al. (1979) Invest. Urol. 17:159-163.
Warhol and Logtine (1985) J. Urol. 134:607.
CA 02232390 1998-03-17
WO97/11172 PCTAUS96/15123
.~u~ : L
(1) GENERAL INFORMATION:
S (i) APPLICANT: W~ROE Sl~K FOUNDATION FOR BIOLOGICAL RESEARCH INC.
(ii) TITLE OF INVENTION: ANTISENSE OLIGONUCL~l'l~ C~EM~THERAPY
FOR BENIGN HYPERPL~SIA OR Q NOE R OF THE PROSTATE
(iii) NUMBER OF ~u~S: 8
(iV) ~VKK~UN~ A~DRESS:
(A) ADDRESSEE: W~LF, GREEWFIFT .n & SACKS, P . c.
(B) ~lK~l: 600 ATLANTIC AVENUE
(C) CITY: BOSTON
(D) STATE: MA
(E) ~VUNl'KY: USA
(F) ZIP: 02210
(v) CQMoeUrER REA~BLE FORM:
(A) MEDrUM TYPE: Floppy disk
(B) CQMPUrER: IBM PC c~m~t;hl~
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFT~RE: PatentIn Release #l.0, Version #l.25
(vi) CU~NT APPLICATICN DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CL~SSIFICATION:
CA 02232390 1998-03-17
WO97/11172 21 PCT~US96/15123
(viii) All~KN~:Y/AGENT INFORMATIQN:
(A) N~ME: TWOMEY, MICH~EL J.
(B) REGISTRATION NUMBER: 38,349
(C) ~K~/DOCKET NUMBER: W0461/7029
s
(ix) TF.T.~C~MUNICATION INFORMATION:
(A) TELEPHONE: 617-720-3500
(B) TELEFAX: 617-720-2441
(2) INFORMATION FOR SEQ ID NO:l:
(i) ~u~ CH~RA~-l~KISTICS:
(A) LENGTH: 5873 base pairs
(B) TYPE: nucleic acid
(C) STRAN~LN~SS: double
(D) TOPOLOGY: l; n~
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYP~l~hllCAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGIN~L SOUR OE:
(A) ORGANISM: HOMO SAPIENS
(G) ~ T- TYPE: LYMPHOID
(H) ~FT-T- LINE: GM 607
(ix) FEATURE:
(A) NAME/KEY: TATA_~lgn~l
(B) LOCATION: 332..338
CA 02232390 l998-03-l7
WO97/11172 22 PCT~US96/15123
(ix) FEATURE:
(A) NAME/KEY: misc_signal
(B) LOCATION: 355..365
(D) OTHER INFORMATION: /note= "TRANSCRIPTIQN~L START
REGION"
(ix) FE~TURE:
(A) NAME/KEY: exon
(B) LOCATION: 401..446
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 1688..1847
~ix) FE~TURE:
(A) NAME/KEY: exon
(B) LOCATION: 34773763
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 39074043
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 5413.. 5568
(ix) F~ATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(401..446, 1688..1847, 3477..3763,
3907... 4043, 5413.. 5568)
CA 02232390 1998-03-17
WO97/11172 23 PCTrUS96/15123
(xi) ~u~ DESCRIPTICN: SEQ ID NO:l:
w~ lAG GCACACT w T CTTZG~aTGC A~AGGATCTA W CACGTGAG G~ ATG 60
A~G~ATCGGG GATCGTACCC ACC('(~'l~'l-l' T~ AT C~'l~C~TG '~ C~l~l~C 120
~ CC~ TAGATGAAGT CTCCATGAGC TACAAGGGCC TGGTGCATCC AG w TGATCT 180
.
AGTAATTGCA GAACAGCAAG TGCTAGCTCT CCCTCCC~-ll CCACAGCTCT ~ W~ lW ~A 240
~l-l~l~C CAGCCTCCAG CAGCATGGGG AG5G~l-1 w TCAGCCTCTG w TGCCAGCA 300
GGGCaaG33C GGAGTCC,T w GGAATGAA~G TTTTATAG w ~l~l~GG w AG5~1~CC~A 360
15 GCCCCA~GCT TACCAACCTGC ACCCGSA~AG ~l~'l~'l'~'ACC ATG TGG GTC CCG GTT 415
Met Trp Val Pro Val
l 5
GTC TTC CTC ACC CTG TCC GTG ACG TGG ATT G GTGAGAaGGa CCAlw~ w 466
20 Val Phe Leu Thr Leu Ser Val Thr Trp Ile
lO 15
GGGGATGCAG GAGaGGGaGC CAGCCCTGAC TGTCA~GCTG AGG~l~l-l-lC CCCCCCAACC 526
25 CAGCACCCCA GCCCAGA~AG w A~l~GC ~ '~l'C~AGC CCCACTTCAA 586
GCCCATACCC CCA~lC~C~l~ CCATATTGCA ACA~l~C~ A CTCCCACAACC A W~l~CCC~C 646
~C~lCC~AC TTACCCCAGA A~lll~llCC CATTTGCCCA GCCA~CTCCC TGCTCCCAGC 706
TGCTTTACTA PPGGGGAAGT lC~l~CAT ~l~C~ l~ GGCTC'AAAAC 766
CA 02232390 l998-03-l7
WO97/11172 PCTAUS96/15123
24
CTCCAAGGAC CTCTCTCAAT GCCA'llw l-l ~ll~ACCG TATCACTGGT CCA'l~lC~l~ 826
AG~CC~'l~AA TCCTATCACA GTCTACTGAC 'l'l'l'l'~C~ATT CAGCTGTGAG TGTCCAACCC 886
S TATCCCAGAG ACCTTGATGC TTGGC~'lC~C AATCTTGCCC TAGGATACCC AGATGCCAAC 946
CAGACACCTC ~'l'l~'l'l'l'~'l' AGCCAGGCTA TCT~GCCTGA GACAACAAAT G~'l'~C~'l~A 1006
~l'~'l~GCAAT GGGACTCTGA GAACTCCTCA 'l'l'CC~'l~ACT ~-ll~CCCCCA GA~l~l'l~AT 1066
TCAGTGGCCC ACA'l'lll'C~'l' TPEGAAAAAC ATGAGCATCC CCAGCC~CAA CTGCCAGCTC 1126
TCTGA~TCCC CAAATCT~CA 'l'~ll'l'l~A AACCTAAAAA CAAAAA3AAA AACA~ATA~A 1186
ACAAAACCAA CTCAGACCAG AA~'l~l'l'l'l'~ TCAA~-l~G A~ll~l~AA ~'l'll'~AAAA 1246
C~ll~l~ll CCA~C~ACTG AACCTCGCCA T~A~GCASTT A'lC~'l~ll' CCTAGCACCC 1306
~ l-lAlCCC~l CAGAATCCAC A~CTTGTACC AA~'l'l'lC~'l' TCTCCCAGTC C~AGACCCCA 1366
AATCACCACA A~GGACCCAA TCCCCAGACT CAAGATATGG 'l~l~ WCGCT ~'l'~'l'l~l~'l'~' 1426
TCCTACCCTG Al~lW~'l' TCA~CTCTGC TCCCAGAGCA TGAAGCCTCT CCACCAGCAC 1486
CAGCCACCAA CCTGCAAACC ~AGGGAAGAT TGACAGAATT CCCAGCClll CCCAGCTCCC 1546
CCTGCCCATG TCCCAGGACT CCC~GCCTTG ~'l'l'~'l'~'l~CC C~l~l~ll TTCA~ACCCA 1606
CATCCTA~AT CC~l'~'l'C~'l'A TCCGAGTCCC CCA~'l'l~CC CTGTC~ACCC TGAll~CC~l 1666
CA 02232390 l998-03-l7
WO97/11172 25 PCTrUS96/15123
GATCTAGCAC ~CC~'l'~'l~'A G GC GCT GCG CCC CTC ATC CTG TCT CGG ATT 1716
Gly Ala Ala Pro Leu Ile Leu Ser Arg Ile
S GTG GGA GGC TGG GAG TGC GAG A~G CAT TCC CAA CCC TGG CAG GTG CTT 1764
Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val Leu
GTG GCC TCT CGT GGC AGG GCA GTC TGC GGC GGT GTT CTG GTG CAC CCC 1812
Val Ala Ser Arg Gly Arg Ala Val CYB Gly Gly Val Leu Val His Pro
CAG TGG GTC CTC ACA GCT GCC CAC TGC ATC AGG AA GTGAGTAGGG 1857
Gln Trp Val Leu Thr Ala Ala His Cys Ile Arg Asn
60 65
GC~lw W'l'C ~l~A~CAG ~ CCC~3~3G~ TPPCAGCTG3 GCAllllCC~ 1917
CAGGATA~CC TCTAAGGCCA G~'1'1W'~AC 'l~AG PLGGAAA3TT ~lW llCAGG 1977
TCAC~TGGGG PEGC~EGG~T GGGGCDGGAC CA~C~l~C' ATG~CTGCCT Gw l~lC~AT 2037
~ C~l~ CTAl~ l~'l~'l'C~CT TTC~TTATGT ~l~llW l~A CTG~ll~w 2097
~ ~l~ACT A~ CT~ '~'l'l'~AGTCT 2157
CCATATCTCC ~C~l'~'l'~'l'~'l' ~l~C~-l~ GGTCC~l~lC TAGCCAGTGT GTCTCACCCT 2217
GTAl~l~l~l GCCA~l~l~ ~l~l~l'~l~ ~'l'~l~'l'~'l'~A C~'l~'l~C~'l'l' ~'l'CC~'l~ACTG 2277
AAC~CAC'GCA ~7~TGGGC ~l~;ACC C~3 GAAGG3Cl-[T GGC GGGCGC 2337
CA 02232390 l99X-03-l7
WO97/11172 PCTGUS96/15123
26
GGTGSCTCAC ACCTGTAATC CC~C~CTTT GSSAGSCCAA GSCAGGTAGA TCACCTGAGS 2397
TCAGGAGTTC GAGACCAGCC TGGCCAACTG GTGAAACCCC ATCTCTACTA A~AATACAAA 2457
S A~ATTAGCCA GS~l~'l~ CGCATGCCTG TAGTCCCAGC TACTCAGGAG CIaAaGaA3G 2517
AGAATTGCAT TGAACCTGGA GGTTG~3GTT GCAGTGAGCC GA~ACCGTGC CACTGCACTC 2577
CA~C~l~l' GACA~AGTGA GA~'l~C~'l' CLAAAAAAAA PAAAAAAAA~ P~4~AA 2637
A~ $~AA ALAAAAaAAA AGG~AGTGTT TTA'lC~l~A 'l~'l~'l~'l~ TATGAGSGTA 2697
TGAGAGaGCC ~l~l~ACTC CAll'~-ll~'l' CC~GGACATC CCTCCACTCT TGGGA3aSAC 2757
AEAGAAGGGC TGGTTCCAGC TGGAGCTGGG PGGGGC~ATT GAGGGAEQAa GAA3GAa~AG 2817
GGGGAAGa~A AACAGGGTAT GGGGGAAAaG AC'C~1~A GCG~AGTGGA GGATACAACC 2877
TTGGGCCTGC AGGCAGSCTA CCTACCCACT TGSAAACCCA CGCCAAAGCC GCATCTACAG 2937
CTGAGCCACT CTGAGGCCTC C~l~C~ ~AC TCAGCTCCAA A~l~l~-l~l~ 2997
C~-llll~l~l CCCACA~l-ll ATCATCCCCC GGAll~l~l CTA~-l'l~'l'l' CTCAl'l~ll~ 3057
25 CTTTa~CTTC CTGCTTCCCT TTCTCATTCA 'l~'l~'l'l'l'~'l'C A~lll~-l~CC 'l~'l'l-l'l~'l'l' 3117
~'l'l~'l'~'l'~'l'C 'l'~'l'l'l'~'l~'l~ GCCCATGTCT ~'l'l'l'~'l'~'lAT ~'l'l'l'~'l~'l'~'l''l'l'l'~'l'l'l'~'l'~ 3177
Al~l~l~lA 'l'lllC~CTC A~ll~lll~ TCA~'l~'l'l'~'l CCCCTCTGCC Cl-l-lCATTCT 3237
CTCTGCC~ll TTA~'l'~'l'l' ~'l'l'l'l'~l''l~'l'l'~'l'~'l'C A~'l'l~l~'~AT CTGCCCTTCA 3297
CA 02232390 l998-03-l7
WO97/11172 27 PCTAUS96/15123
~l~l~ACA CTG~ C' CCA~ TGGCCTG~AC ~ l~C 3357
CAA~C~-~ ~CT ~ ~l~llll~GA G~-l~ TG~-lC~l~l~ 3417
S ~l~C~ C~l~AT CA~l~C~ CT CCTCATTCCT G~l~l~CTT ~l~AGC 3477
A~A AGC GTG ATC TTG CTG GGT C'GG CAC AGC CTG TTT CAT CCT G~A GAC 3525
Lys Ser Val Ile Leu Leu Gly Arg His Ser Leu Phe His Pro Glu Asp
ACA G5C CAG GTA TTT CAG GTC AGC CAC AGC TTC CCA CAC CC'G CTC TAC 3573
Thr Gly Gln Val Phe Gln Val Ser His Ser Phe Pro His Pro Leu Tyr
100
GAT ATG AGC CTC CTG A~G AAT CGA TTC CTC AGG CCA GGT GAT GAC TCC 3621
Asp Met Ser Leu Leu Lys Asn Ary Phe Leu Arg Pro Gly Asp Asp Ser
105 110 115
AGC CAC GAC CTC ATG CTG CTC C'GC CTG TCA GAG CCT GCC GAG CTC ACG 3669
Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Glu Leu Thr
120 125 130
GAT GCT GTG A~G GTC ATG GAC CTG CCC ACC CAG GAG CCA GCA CTG GGG 3717
Asp Ala Val Lys Val Met Asp Leu Pro Thr Gln Glu Pro Ala Leu Gly
135 140 145
ACC ACC TGC TAC GCC TCA G5C TGG G5C AGC ATT G~A CCA GAG GAG T 3763
Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu Glu
150 155 160
GTACGCCTGG GCCAGATGGT GC~GCCG5GA GCCCAGATGC CTGGGTCTGA G5GAGGAGGG 3823
CA 02232390 1998-03-l7
WO97/11172 PCT~US96/15123 28
GACAGGACTC CTG W~l~l~A GGG~3GA33G CCAAGGAACC A~l~l~ CAGCCCACAA 3883
CA~l~lllll GCCTGGCCCG TAG TC TTG ACC CCA A~G A~A ~ CAG TGT 3932
Phe Leu Thr Pro Lys Lys Leu Gln Cys
165 170
GTG GAC CTC CAT GTT ATT TCC AAT GAC GTG TGT GCG CAA GTT CAC CCT 3980
Val Asp Leu His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro
175 180 185
CAG A~G GTG ACC A~G TTC ATG CTG TGT GCT G~A CGC TGG ACA GGG GGC 4028
Gln Lys Val Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly
190 195 200 205
A~A AGC ACC TGC TCG GTGAGTCATC CCTACTCCCA AGATCTIGAG GSA~AGGTGA 4083
Lys Ser Thr Cys Ser
210
GT~GG~CCTT AATTCTGGGC l~l~l~G AA~CCAACAA ~ '~l~CC ~l~C'C~l~lC 4143
CCC~3CT3TA GC~ATGCC~C ~l~CC~l~l CTCATCTCAT TCC~lC~lLC C~-l~l-l~l-ll 4203
GA~l~C~l~A AGGC~ATAGG TTATTCTTAC AGCACAACTC Al~l~ll~l G~TTCAGCA 4263
CAC~GTTACT AGGCACCTGC TATGCACCCA GCA~l~C~L AGAG~l~ W ACATAGCAGT 4323
G~ACA~ACAG AGAGCAGCCC ~'l~'l'l'~'l~ TAGCCCCCAA GCC~GTGAGG GGC~Ca3GC~ 4383
G5A~C~GGGA CCACAACACA GA~AAGCTGG A~ l~AG GAGGTGATCA GS~L~l~ 4443
G~3G3~GA~G ~l~AG TGTGACTGGG A~GaGAcATc CTGCAGA~G TGGGA~TGAG 4503
CA 02232390 1998-03-17
W O 97/11172 PCTAJS96/lS123
29
CA~ACACCTG CG~P~~3G~G CC~ GCGGCACCTG GG~ GGGA~CAGCA 4563
TCTGGCCAGG C~'1~X~a~ GGGGCCTAGA GGGCGTCAGG AGCAGAGAGG AGGTTGCCTG 4623
GCq~ GA AGGA'1'~G CAGGGTGCGA GAGGGA~CA~ AGGA~C~1~ cT~K~Y~aGcc 4683
TCA~ C CA5~ GA CACT~FrrT C~ 1~AGGA GTCAGGAACT GTGGATGGTG 4743
CqYK~}~a~ GCAGGACAGG ~G~'l~'l'~A G~ C~AGA GGC~X~C~G GCCTCCTATG 4803
GGATCAGACT GC~ 3GG AE~YK}Y3C~3 GGA1~1W AG GGAGTGATGA TY~KK~GAC 4863
~'l W'~l W CTCCAGGCAT 1~1CC~ACC TGGGCCC11A CCC~GCCTCC CTCACAGGCT 4923
CCT~GCCCTC A~1~1~ CCC CTCCACTCCA 'l'l~l'C~CCT ACCCACAGTG GGTCATTCTG 4983
ATCACCG~AC TGACCATGCC AGCCCTGCCG A~1W 1~1~ ATGG~1CC~1~ AGTGC~1 W 5043
P~F~U~3TG TCTAGTCAGA GAGTAGTCCT GGAAGGTGGC ~1~1~1~GG AGCCACGGGG 5103
ACAGCATCCT GCAGATGGTC CTGG~11~ TCCCACCGAC ~1~1~1ACAA GGA~-1~1~-1 5163
CGTGGACCCT ~1~1~CA CALY~ DGG ACCCT~AAGT CCCTTCCTAC CGGCCAGGAC 5223
TGGAGCCCCT A~C~l~'l~'l' TGGAATCCCT GCCCAC~-l-lC 'l-l'~l~AAGT oGG~'l'~'l~A 5283
GACAlll~-lC 'l'~'l'l'~'l'l'C~'A AALCTGGGAA CT~CTATCTG TTATCTGCCT GTCCAGGTCT 5343
GAAAGATAGG ATTGCCCA~G CAGAAACTGG GACTGACCTA TCTCACTCTC TCCCTGCTTT 5403
TACCC'llAG GGT GAT TCT GGG GGC CCA CTT GTC TGT AAT GGT GTG CTT 5451
CA 02232390 1998-03-17
WO97/11172 PCT~US96/15123
Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu
215 220
CAA GGT ATC ACG TCA TGG G5C AGT GAA CCA TGT GCC CTG CCC GAA AGG 5499
Gln Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg
225 230 235
CCT TCC CTG TAC ACC A~G GTG GTG CAT TAC CGG A~G TGG ATC A~G GAC 5547
Pro Ser Leu Tyr Thr Lys Val Val His Tyr Ary Lys Trp Ile Lys Asp
10 240 245 250 255
ACC ATC GTG GCC AAC CCC TGA~CACCCC TATC~AGTCC CTATTGTAGT 5595
Thr Ile Val Ala Asn Pro
260
A~ACTTGGAA C~ll~AAAT GACCA~5CCA AGACTCAAGC ~lCC~AGTT CTACTGACCT 5655
l-l~TCCTTAG GTGTGA~GTC CA~ll~l'AGGPPA~EA~ ATCAGCAGAC ACAGGTGTAG 5715
20 ACCAGAGTGT TTCTTA~ATG GTGTAATTTT ~ ~lC~l~ AATACT~5CC 5775
ATGCCTGGAG ACATATCACT CAAll-l~l~l~ GAGGACACA5 TTAGGATGGG ~1~1~1~1~1~ 5835
TAlll~l~ ATACA~AGAT GA~AG~3GGG TGGGATCC 5873
(2) INFORMATICN FOR SEQ ID N~:2:
(i) SEQUENOE CH~RACTERISTICS:
(A) LEN~TH: 776 base pairs
(B) TYPE: nucleic acid
CA 02232390 l998-03-l7
WO97/11172 31 PCTrUS96/15123
(C) STRANDEDWESS: single
(D) TOPOLOGY: 1;n~r
( ii ) ~T .T~lT .T~ TYPE: mRNA
s
(iii) HYP~l~l~lCAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGIN~L SCUR OE :
(A) ORGANISM: RATTUS NORVEGICUS
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 41... 574
(ix) FEATURE:
(A) NAME/KEY: sig peptide
(B) LOCATION: 41..91
(ix) FEATURE:
(A) NAME/KEY: mat peptide
(B) LOCATION: 92..571
(xi) SEQ~EN OE DESCRIPTION: SEQ ID NO:3:
-l~'l~AGTG AGGTCC~AT ACCTACAGAG CTCACACACG ATG AGG GTC ATC CTC 55
Met Arg Val Ile Leu
-17 -15
CA 02232390 l998-03-l7
WO97/11172 PCT~US96/15123
32
CTC CTG CTC ACA CTG GAT GTG CTA GGT GTC TCC AGT ATG ATG ACA GAC 103
Leu Leu Leu Thr Leu Asp Val Leu Gly Val Ser Ser Met Met Thr Asp
-10 -5
5 A~G AAT CTC A~A A~G A~ ATT GAA G5G AAT TGG AGA ACC GTT TAC TTA 151
Lys Asn Leu Lys Lys Lys Ile Glu Gly Asn Trp Arg Thr Val Tyr Leu
GCT GCC AGT AGC GTG GAG AAG ATA AAT G~A GGC TCA CCA TTG AGG ACC 199
Ala Ala Ser Ser Val Glu Lys Ile Asn Glu Gly Ser Pro Leu Arg Thr
TAC TTC CGT CGC ATT GAG TGT GGG A~G AGA TGC A~C CGA ATC AAT CTC 247
Tyr Phe Arg Arg Ile Glu Cys Gly Lys Arg Cys Asn Arg Ile Asn Leu
40 45 50
TAC TTT TAT ATT A~G A~A GGG GCC A~G TGC CAA CAG l~ A~A ATC GTG 295
Tyr Phe Tyr Ile Lys Lys Gly Ala Lys Cys Gln Gln Phe Lys Ile Val
GGA AGG AGA TCC CAA GAC GTT TAC TAT GCA A~G TAT GAA GGG AGC ACG 343
Gly Arg Arg Ser Gln Asp Val Tyr Tyr Ala Lys Tyr Glu Gly Ser Thr
25 GCA TTC ATG TTA AAG ACA GTG AAT GAG AAG ATA TTG CTG TTT GAT TAT 391
Ala Phe Met Leu Lys Thr Val Asn Glu Lys Ile Leu Leu Phe Asp Tyr
100
TTT AAC AGA AAC AGA AGA AAT GAC GTT ACA CGA GTG GCT GGA GTT TTG 439
Phe Asn Arg Asn Arg Arg Asn Asp Val Thr Arg Val Ala Gly Val Leu
105 110 115
CA 02232390 l998-03-l7
WO97/11172 PCT~US96/15123
GCG A~A GGC AGG CAA CTG ACT A~G GAT GAG ATG ACA GAA TAC ATG A~C 487
Ala Lys Gly Arg Gln Leu Thr Lys Asp Glu Met Thr Glu Tyr Met Asn
120 125 130
5 TTC GTG GAA GAA ATG GGC ATT GAG GAT GAG AAT GTA Q A CGT GTC ATG 535
Phe Val Glu Glu Met Gly Ile Glu Asp Glu Asn Val Gln Arg Val Met
135 140 145
GAC ACA GAC ACC TGT CCA AAC A~G ATC AGA ATT AGA TGACATCAGG 581
10 Asp Thr Asp Thr Cys Pro Asn Lys Ile Arg Ile Arg
150 155 160
A~llllC~AC TATAll~llC CTGGAACCTG A~ACAT Q AT ATGA~GATGA AGCAAT~-lll 641
15 ~ ll~AGAT CATATCl-l~CC TAlll~l~ AAATTACAAT ~ ATA~lll~lC 701
lllCATTCAT A~lll~C~AT GTTCTAATTG GTATTAGTAC Al~-lll~AT GlllAAATA~ 761
ATCTAlllCA CTTGC 776
(2) INFORMATION FOR SEQ ID NO:3:
(i) ~u~ CHARALl~KISTICS:
(A) LEN~TH: 27 base pairs
(B) TYPE: ml~l ~; C acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: 1;n~
(ii) MOLEC~LE TYPE: cDNA
CA 02232390 1998-03-17
WO97/11172 PCT~US96/15123
34
(iii) HYPVl~l~lCAL: NO
(iv) ANTI-SENSE: YES
S (vi) ORIGINAL SC~R OE :
(A) ORGANISM: SYNIHETIC OLIGCNUCL~ lv~
(ix) FEATURE:
(A) NAME/KEY: misc_~eature
10 (B) LOCATION: l.. 27
(D) OTHER INFORMATIQN: /note= "ANTISENSE TO POSITIONS
92-118 OF SEQ ID NO.: 2."
(xi) ~U~ DESCRIPTION: SEQ ID NO:3:
~lllll~A ~ 1~ TCATCAT 27
(2) INFORMATION FOR SEQ ID NO:4:
(i) ~u~N~ CH~RACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: 1 ;n~r~
(ii) MOLEC~LE TYPE: cDNA
(iii) EYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
CA 02232390 1998-03-17
WO97/11172 PCTAJS96/15123
(vi) ORIGIN~L SOUR OE :
(A) ORGANISM: SYNTHETIC OLIG~NUCL~ll~
(ix) FEATURE:
S (A) NAME/KEY: misc_feature
(B) LOCATION: 1..24
(D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS
76-99 OF SEQ ID NO.: 2."
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:4:
GTCATCATAC TaG~aA~CC TAGC 24
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nllcl ~; C acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: 1; n~
(ii) MOLECULE TYPE: cDNA
(iii) HYP~ CAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGIN~L SOUR OE:
(A) ORGANISM: ~YN'l'~'l'lC OLIGONUCL~ll~
CA 02232390 1998-03-17
WO97/11172 36 PCT~US96/15123
(ix) FEATURE:
(A) NAME/KEY: misc_~ea~ure
(B) LOCATIOW: 1..21
(D) OIHER INFORMATION: /note= "ANTISENSE TO POSITIONS
80-l00 OF SEQ ID NO.: 2."
(xi) ~U~N~ DESCRIPTION: SEQ ID NO:5:
10 TGTCATCATA CTGGAGACAC ~l~l~AGT 29
(2) INFORM~TION FOR SEQ ID NO:6:
(i) SEQUENOE CH~RA~l~ISTICS:
(A) T ~r7T~ 29 base pairs
(B) TYPE: nl]~l ~; C acid
(C) STRANDE~NESS: single
(D) TOPOLOGY: linear
(ii) ~T ~~lT .F. TYPE: cDN~
(iii) ~Y~l~l~lCAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGIN~L SOUROE :
(A) ORGANISM: SYNTHETIC OLIG~NUCL~u
(ix) FEATURE:
(A) NAME/KEY: misc_~eature
(B) LOCATIOW: 1..21
CA 02232390 1998-03-17
WO97/11172 PCT~US96/15123
(D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS
92-112 OF SEQ ID NO.: 2"
s
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO:6:
GA~ ~ATCA TTGAC~AC 29
(2) INFORMATION FOR SEQ ID NO:7:
(i) ~U~ CHARALl~KISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRA~Lw~SS: single
(D) TOPOL~Y: 1i n~
(ii) MOLECULE TYPE: cDNA
(iii) HYPOl~llC~L: NO
(iv) ANTI-SENSE: YES
(vi) ORIGIN~L SOUROE:
(A) ORG~NISM: SYNTHETIC OLIGCWUCL~
(ix) FEATURE:
(A) NAME/KEY: misc_feature
J (B) LOC~TIQN: 1.. 27
(D) OTHER INFORMATIQN: /note= "ANTISENSE TO POSITIONS
401-427 OF SEQ ID NO.: l."
CA 02232390 1998-03-17
WO97/11172 PCTAJS96/15123
38
(xi) ~U~N~ DESCRIPTION: SEQ ID NO:7:
GGTGA3G~A~ ACA~r~.~ CCCACAT 27
(2) INFORMATION FOR SEO ID NO:8:
(i) SEQUENOE CH~RACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRAN~LN~SS: single
(D) TOPOLOGY: 1; n~
(ii) ~T~ T~F TYPE: cDNA
(iii) HYP~~ CAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOUROE :
(A) OR~ANISM: SYNTHETIC OLIGONU~I~ll~
(ix) FEATURE:
(A) NAME/KEY: misc_~eature
25 (B) LOCATIQN: 1.. 27
(D) OTHER INFORMATION: /note= "ANTISENSE TO POSITIONS
384-410 OF SEQ ID NO.: l."
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:8:
GGACCCACAT GGTGACAC~ ~1~ 27
