U.S. patent application number 11/827995 was filed with the patent office on 2008-04-24 for inhibitory oligonucleotides targeted to bcl-2.
Invention is credited to Zhidong Chen, Richard Koehn, Ramesh Prakash, Duane E. Ruffner.
Application Number | 20080096835 11/827995 |
Document ID | / |
Family ID | 32326325 |
Filed Date | 2008-04-24 |
United States Patent
Application |
20080096835 |
Kind Code |
A1 |
Chen; Zhidong ; et
al. |
April 24, 2008 |
Inhibitory oligonucleotides targeted to BCL-2
Abstract
Inhibitory oligonucleotides are disclosed which are targeted to
three specific target regions and subsequences of the target
regions found on nucleic acids encoding Bcl-2. These inhibitory
oligonucleotides are generally of from about 8 to about 50
nucleotides in length. Specific preferred oligonucleotides are
disclosed. The oligonucleotides of the invention may be
incorporated into compositions such as pharmaceutical compositions,
and may be used in methods for inhibiting the expression of Bcl-2
in a cell or tissue, methods for treating conditions susceptible to
modulation of Bcl-2 expression in an organism, and methods for
detecting nucleic acid encoding BCL-2.
Inventors: |
Chen; Zhidong; (Salt Lake
City, UT) ; Ruffner; Duane E.; (Salt Lake City,
UT) ; Prakash; Ramesh; (Salt Lake City, UT) ;
Koehn; Richard; (Salt Lake City, UT) |
Correspondence
Address: |
KENYON & KENYON LLP
ONE BROADWAY
NEW YORK
NY
10004
US
|
Family ID: |
32326325 |
Appl. No.: |
11/827995 |
Filed: |
July 12, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10714310 |
Nov 14, 2003 |
7256284 |
|
|
11827995 |
Jul 12, 2007 |
|
|
|
60426269 |
Nov 14, 2002 |
|
|
|
Current U.S.
Class: |
514/44R ;
536/23.1 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 43/00 20180101; C12N 2310/11 20130101; A61P 29/00 20180101;
C12N 2310/3517 20130101; A61P 35/00 20180101; A61P 31/00 20180101;
C12N 15/1135 20130101; C12N 2310/315 20130101 |
Class at
Publication: |
514/044 ;
536/023.1 |
International
Class: |
A61K 31/711 20060101
A61K031/711; C07H 21/02 20060101 C07H021/02; C07H 21/04 20060101
C07H021/04 |
Claims
1. An isolated oligonucleotide comprising a sequence of at least 8
contiguous nucleobases which is substantially identical or
complementary to at least a portion of SEQ ID NO: 19 or SEQ ID NO:
19 wherein U is substituted for T.
2. The oligonucleotide of claim 1 wherein the sequence is
substantially identical or complementary to SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ
ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
24, SEQ ID NO: 25 or an any of the foregoing SEQ ID NOs. wherein U
is substituted for T.
3. The oligonucleotide of claim 1 wherein the substantially
identical or complementary sequence is about 10-26 nucleobases in
length.
4. The oligonucleotide of claim 1 which is an RNA
oligonucleotide.
5. The oligonucleotide of claim 1 which comprises a non-naturally
occurring nucleobase, sugar or internucleotide linkage.
6. The oligonucleotide of claim 5 which comprises a
phosphorothioate internucleotide linkage.
7. A composition comprising the oligonucleotide of claim 1 and a
pharmaceutically acceptable carrier, diluent or adjuvant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
10/714,310, filed Nov. 14, 2003, which claims the benefit of U.S.
Provisional Application No. 60/426,269, filed Nov. 14, 2002, hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to agents and
nucleic acid targets for regulating gene expression. More
specifically, the present invention relates to compounds,
compositions, and methods for regulation of the expression of
nucleic acids encoding Bcl-2.
[0004] 2. Technical Background
[0005] The genetic blueprint of mankind is written and stored in
the chromosomes found in the nuclei of somatic cells. These
chromosomes are each made up of a single molecule of DNA. DNA is a
double-stranded polymer compound whose strands are chains of the
nucleotide subunits adenine, thymine, cytosine, and guanine. These
chains are held to each other by hydrogen bonds formed between
complementary nucleotides on the chains. Adenine (A) forms hydrogen
bonds with thymine (T); and guanine (G) forms hydrogen bonds with
cytosine (C). Contiguous sets of three of these nucleobases in the
nucleotide strands code for individual amino acids. Together, the
nucleotide chains of DNA code for all of the various proteins
needed to build, maintain, and fuel the human body.
[0006] Proteins are translated from transcribed copies of DNA
called messenger RNA. Messenger RNA is made when DNA partially
unwinds, allowing the sequence of the DNA to be copied and
transcribed by cellular machinery. Messenger RNA is made of similar
components as DNA, with the substitution of uracil for thymine and
the substitution of ribose for deoxyribose in all nucleotides. Once
the transcription of a messenger RNA is completed, it is
transported from the nucleus to the cytosol. There, the mRNA is
engaged by the enzymes that are the protein-manufacturing machinery
of the cell. The enzymes and other associated molecules making up
this machinery bind to the mRNA and begin to interpret the code of
the messenger RNA. As they do so, they also assemble and bind the
amino acids encoded into chains to produce the protein coded for by
the original DNA.
[0007] Illnesses and disease states such as some cancers have been
linked to the over-, under-, or mis-production of specific
proteins. In some of these cases, a defect present in the gene
causes the production of messenger RNAs that code for a
nonfunctional or inefficient protein. Additionally, some diseases
may be controlled by increasing or decreasing the normal production
of a protein. Specifically, it is thought that in some cancers, if
the gene coding for a specific protein could be regulated, the
cancer would abate, or would become more susceptible to treatment.
As a result of this, much research has been conducted to find a
method of modulating the activity of a gene. In one specific study,
it was determined that down-regulation of Bcl-2 production in
prostate cancer cells inhibited their growth and rendered them
susceptible to adriamycin-induced apoptosis. Shi et al., Cancer
Biother. Radiopharm., 16(5):421-9 (October 2001).
[0008] One technology used for regulating the expression of a gene
is antisense technology. Antisense technology controls the
production of a protein by binding a molecule to the nucleic acid
coding for the protein. Generally this molecule is a short length
of DNA or RNA commonly referred to as an antisense oligonucleotide.
These oligonucleotides are complementary to a segment of the
nucleic acid. In use, these antisense oligonucleotides are
administered to a cell or tissue desired to be treated. The
oligonucleotides are taken into the cell where they associate with
and bind to a region of the nucleic acid encoding the protein to
which they are complementary. This binding prevents normal
interaction of the nucleic acid with cellular machinery such as RNA
translation enzymes or induces the degradation of the message RNA.
This inhibits or prevents proper translation of the message
RNA.
[0009] Antisense is regarded by many as a powerful technology since
the antisense oligonucleotides used may be carefully targeted to
specific regions on the nucleic acids, thus preventing interaction
of the oligonucleotides with other molecules not desired to be
inhibited or activated. These specific regions of the selected
nucleic acids are often referred to as target sequences. Because
antisense oligonucleotides may be so carefully targeted to these
target sequences, antisense oligonucleotides may be used to provide
compositions, such as drugs, that have near-absolute specificity,
high efficacy, low toxicity, and few side effects.
[0010] These benefits are overshadowed, however, by the difficulty
involved in locating effective antisense oligonucleotides for use
with a gene. Despite the fact that generally there are a large
number of potential oligonucleotides available for each individual
gene, different antisense oligonucleotides have different effects
on gene expression. This has been shown to be due at least in part
to the final folded structure of the nucleic acid which may block
access to some regions of the nucleic acid sequence, thus rendering
some oligonucleotides ineffective. Further, the work of
characterizing effective sites must be repeated for each individual
gene desired to be targeted. This process is generally a long and
expensive one. Many disease states, including cancer, stand to
benefit from successful antisense therapies, but oligonucleotides
useful with specific genes are elusive.
[0011] Bcl-2 is an inner mitochondrial membrane protein that has
been shown to block apoptotic cell death in some specific cell
types. The expression of Bcl-2 has been shown to be involved in
apoptosis in the thymus (Kanavaros et al., Histol. Histopathol.
16(4):1005-12 (October 2001). Bcl-2 has also been shown to play a
role in prostatic cancers, and has been specifically linked to
aggressive tumors common in specific racial groups. Shi et al.,
Cancer Biother. Radiopharm., 16(5):421-9 (October 2001); Slothower,
Study Suggests Bcl-2 Gene as a Cause for Aggressive Prostate Cancer
in African American Men, U.C. Davis Med. Ctr., (May 1998). As
briefly noted above, a study in which Bcl-2 production was
down-regulated in prostate cancer cells showed inhibition of cell
growth and increased sensitivity to treatments designed to induce
apoptosis. Shi et al., Biother. Radiopharm., 16(5):421-9 (October
2001).
[0012] Accordingly, it would be an advancement in the art to
provide inhibitory oligonucleotide compounds configured to bind to,
and consequently, to modulate the activity of nucleic acids
encoding proteins which play a role in diseases such as cancer. It
would be a further advancement to provide effective target sites
for Bcl-2 gene regulation. It would be an advancement in the art to
provide oligonucleotides complementary to effective antisense
target regions of a nucleic acid encoding Bcl-2. It would be a
further benefit in the art to provide compositions such as
medications, including such oligonucleotides. Finally, it would be
an improvement in the art to provide methods of using such
oligonucleotides and compositions.
[0013] Such oligonucleotides, target regions, compositions
including such oligonucleotides, and methods of their use are
disclosed herein.
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention is directed to oligonucleotides that
are targeted to nucleic acids which encode Bcl-2. The compounds are
designed to be complementary to at least a part of a target region
of a nucleic acid encoding Bcl-2, and as a result, to modulate the
expression of Bcl-2.
[0015] The invention first encompasses compounds such as
oligonucleotides configured to hybridize with at least a portion of
an oligonucleotide encoding Bcl-2. One such oligonucleotide
encoding Bcl-2 is the oligonucleotide of SEQ ID NO: 18. A sequence
variant of a Bcl-2 cDNA is shown in SEQ ID NO: 35. Some of these
compounds are oligonucleotides of between about 8 and about 30
nucleobases in length having at least 8 contiguous nucleobases
complementary to at least a portion of SEQ ID NO: 18. As used
herein, the term "nucleobase" refers to adenine (A), cytosine (C),
guanine (G), thymine (T) and uracil (U); their post-replicationally
or post-transcriptionally modified derivatives; and other bases
suitable for inclusion in a nucleic acid. Inhibitory
oligonucleotides of the invention are presented in the attached
Sequence Listing as DNA sequences for convenience. It is to be
understood, however, that the corresponding RNA inhibitory
sequences, routinely obtained by substituting "U" for "T" in the
sequences set forth in the Sequence Listing, are included in the
invention.
[0016] In specific embodiments, the oligonucleotides of the
invention include at least about 8 contiguous nucleotides
complementary to at least a portion of SEQ ID NO: 19 or SEQ ID NO:
36, preferably complementary to at least a portion of SEQ ID NO:
15. Some such oligonucleotides have a sequence selected from the
group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14, SEQ
ID NO: 22, SEQ ID NO: 23, SEQ ID NO:24 and SEQ ID NO:25.
[0017] In other embodiments, the oligonucleotides of the invention
include at least about 8 contiguous nucleotides complementary to at
least a portion of SEQ ID NO: 20 or SEQ ID NO: 37, preferably
complementary to at least a portion of SEQ ID NO: 16. Some such
oligonucleotides have a sequence selected from the group comprising
SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID
NO: 28, SEQ ID NO: 29 and SEQ ID NO:30.
[0018] In other embodiments, the oligonucleotides of the invention
include at least about 8 contiguous nucleotides complementary to at
least a portion of SEQ ID NO: 21, preferably complementary to at
least a portion of SEQ ID NO: 17. Some such oligonucleotides have a
sequence selected from the group comprising SEQ ID NO: 10, SEQ ID
NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 32,
SEQ ID NO: 33 and SEQ ID NO: 34.
[0019] The oligonucleotides of the invention may include features
or components such as modified internucleotide linkages, modified
sugar moieties, and modified nucleobases. The oligonucleotides may
also contain nucleotides from DNA or RNA.
[0020] The invention further includes compositions such as
pharmaceuticals including the oligonucleotide compounds disclosed
above that are targeted to any of the target regions of the
invention found in SEQ ID NO: 18 or SEQ ID NO: 35, including the
regions of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 36 and SEQ ID NO: 37.
Such compositions may include additional components such as
pharmaceutically-acceptable carriers, diluents or adjuvants.
[0021] The invention also includes methods of inhibiting the
expression of Bcl-2 in a cell or tissue. Such methods include the
step of contacting the cell or tissue with a composition made with
the oligonucleotides of the invention. Such oligonucleotides may
include compounds comprising a segment of from about 8 to about 30
nucleobases in length of a sequence chosen from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID
NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,
SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID
NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,
SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID
NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32,
SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36 and SEQ ID NO: 37.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] A more particular description of the invention briefly
described above will be rendered by reference to specific
embodiments thereof which are illustrated in the appended drawings.
These drawings depict only typical embodiments of the invention and
are not therefore to be considered to be limiting of its scope. The
invention will be described and explained with additional
specificity and detail through the use of the accompanying drawings
and detailed description, in which:
[0023] FIG. 1 is an anti-Bcl-2 antisense target map showing the
inventive oligonucleotides and the corresponding region of a
portion of the Bcl-2 cDNA from which they are derived (SEQ ID NO:
38).
[0024] FIG. 2 shows the results of Example 2.
[0025] FIG. 3 shows the results of Example 3.
DETAILED DESCRIPTION
[0026] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of virology,
microbiology, molecular biology, and recombinant DNA techniques
within the skill of the art. Such techniques are fully explained in
the literature. See, e.g., Sambrook, et al., Molecular Cloning: A
Laboratory Manual (Current Edition); DNA Cloning: A Practical
Approach, vol. I & II (D. Glover, ed.); Oligonucleotide
Synthesis (N. Gait, ed., Current Edition); Nucleic Acid
Hybridization (B. Hames & S. Higgins, eds., Current Edition);
Transcription and Translation (B. Hames & S. Higgins, eds.,
Current Edition); CRC Handbook of Parvoviruses, vol. I & II (P.
Tijessen, ed.); Fundamental Virology, 2nd Edition, vol. I & II
(B. N. Fields & D. M. Knipe, eds.).
[0027] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety. As used in this specification and the
appended claims, the singular forms "a," "and," and "the" include
plural references unless the content clearly dictates
otherwise.
[0028] The present invention relates to oligomeric compounds for
modulating the function of nucleic acid molecules encoding Bcl-2.
More specifically, the invention relates to oligomeric compounds
such as inhibitory oligonucleotides that are configured to interact
with nucleic acids encoding Bcl-2. The invention further includes
compositions comprising such oligonucleotides, including
pharmaceutical compounds, and methods for their use. The invention
additionally includes methods of treating diseases that respond to
the modulation of Bcl-2 such as several cancers.
1. DEFINITIONS
[0029] The following terms will be used in describing the compounds
and methods of the instant invention. They are intended to be
defined as indicated hereafter.
[0030] As used herein, the terms "oligonucleotide" and "nucleic
acid" denote polynucleotides-polymers of nucleotides. Further, as
used herein, the terms "target nucleic acid" and "nucleic acid
encoding Bcl-2" include polynucleotides having at least a portion
of the sense or antisense code for Bcl-2. DNA encoding Bcl-2 is
thus included, as is RNA such as pre-mRNA and mRNA, cDNA, and
hybrid nucleic acids such as artificial sequences having at least a
portion of the sequence of Bcl-2. Herein, the terms "nucleic acid,"
"target nucleic acid," and "nucleic acids encoding Bcl-2" also
include sequences having any of the known base analogs of DNA and
RNA such as, but not limited to 4-acetylcytosine,
8-hydroxy-N-6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methyl guanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxy-aminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, 2,6-diaminopurine, and
2'-modified analogs such as, but not limited to O-methyl, amino-,
and fluoro-modified analogs.
[0031] In the context of this invention, the term
"oligonucleotides" refers to an oligomer or polymer of ribonucleic
acid (RNA) or deoxyribonucleic acid (DNA) or analogs thereof. This
term includes oligonucleotides composed of naturally-occurring
nucleobases, sugars and internucleotide (or "backbone") linkages,
as well as oligonucleotides having non-naturally occurring portions
with similar function.
[0032] Herein, the term "inhibitory oligonucleotide" denotes an
oligonucleotide having a sequence that enables the oligonucleotide
to interact with a selected portion of a nucleic acid. This
interaction is often sufficient to result in the disruption of the
function of the nucleic acid. The oligonucleotides of the invention
may be either RNA or DNA and have a sequence that is substantially
complementary to at least a segment of the selected portion of the
nucleic acid. As such, the oligonucleotide may have a substantially
sense sequence or a substantially antisense sequence. Such
oligonucleotides may disrupt the function of the nucleic acid by
specifically hybridizing with it. Some such oligonucleotides may
specifically hybridize to the selected portion of the nucleic acid.
Additionally, the inhibitory oligonucleotide may have a sequence
that is substantially identical to that of the selected portion of
the nucleic acid.
[0033] The term "antisense oligonucleotide" is used herein to
denote an oligonucleotide which is complementary to, and thus has
the capacity to specifically hybridize with, a nucleic acid. This
is especially used herein to refer to oligonucleotides whose
binding modulates the normal activity or function of the target
nucleic acid. The modulation of nucleic acid activity caused by
such oligonucleotides is broadly termed "antisense" technology.
Antisense oligonucleotides may be either RNA or DNA.
[0034] The inhibitory oligonucleotides of the present invention
include antisense oligonucleotides. The antisense oligonucleotides
of the invention may be equal in size to the entire target sequence
to which they are targeted. Alternatively, such oligonucleotides
may be from about 8 to about 50 nucleobases in length (i.e. from
about 8 to about 50 linked nucleosides). Still further, the
oligonucleotide compounds may be antisense oligonucleotides of from
about 10 to about 30 nucleobases in length. The antisense
oligonucleotide compounds of the invention may be either RNA or DNA
and include without limitation ribozymes, external guide sequences
(EGS), oligozymes, other short catalytic RNAs and other catalytic
oligonucleotides, or other short RNAs such as siRNA, miRNA, and
shRNA, etc., which are configured to hybridize to the target
nucleic acid and modulate its expression or integrity.
[0035] The term "complementary," as used herein, refers to the
capacity of two nucleotides to pair precisely with each other. This
is often termed "Watson-Crick pairing." This term may also be used
to refer to oligonucleotides which exhibit the ability of pairing
precisely with each other. For example, if the nucleotides located
at a certain position on two oligonucleotides are capable of
hydrogen bonding, then the oligonucleotides are considered to be
complementary to each other at that position. The oligonucleotides
and the DNA or RNA themselves are complementary to each other when
a sufficient number of corresponding positions in each molecule are
occupied by nucleotides which can hydrogen bond with each other.
Thus, "specifically hybridizable" and "complementary" are terms
which are used to indicate a sufficient degree of complementarity
or precise paring such that stable and specific binding may occur
between the oligonucleotides and the DNA or RNA target.
[0036] It is understood in the art that the sequence of an
inhibitory oligonucleotide compound need not be 100 percent
complementary to that of its target nucleic acid to be specifically
hybridizable. An inhibitory oligonucleotide compound is
specifically hybridizable when binding of the compound to the
target DNA or RNA molecule interferes with the normal function of
the target DNA or RNA. A sufficient degree of complementarity
prevents non-specific binding of the inhibitory oligonucleotide
compound to nontarget sequences under conditions in which specific
binding is desired, i.e. under physiological conditions in the case
of in vivo assays or therapeutic treatment, and in the case of in
vitro assays, under conditions in which the assays are
performed.
[0037] As is known in the art, a nucleoside is a combination of a
base and a sugar. The base portion of the nucleoside is normally a
heterocyclic base. The two most common classes of heterocyclic
bases are purines and pyrimidines.
[0038] Nucleotides are nucleosides that additionally include a
phosphate group linked to the sugar portion of the nucleoside. For
those nucleosides that include a pentafuranosyl sugar, the
phosphate group can be linked to either the 2', 3', or 5' hydroxyl
moiety of the sugar. In forming oligonucleotides, the phosphate
groups link adjacent nucleosides to one another to a form a linear
polymer. The ends of such linear polymers can also be joined, thus
forming a circular structure. Within the oligonucleotide structure,
the phosphate groups form the internucleotide backbone of the
oligonucleotide. The normal linkage or backbone of RNA and DNA is a
3' to 5' phosphodiester linkage.
[0039] Examples of compounds useful in this invention include
oligonucleotides containing modified backbones or non-natural
internucleotide linkages. Possible modified oligonucleotide
backbones include, for example, phosphorothioates, chiral
phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates, phosphinates, phosphoramidates
including 3'-amino phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, selenophosphates, and
borano-phosphates having normal 3'-5' linkages, as well as their
2'-5'-linked analogs. These modified oligonucleotides may have
inverted polarity, where one or more of the internucleotide
linkages is a 3' to 3', 5' to 5', or 2' to 2' linkage.
Oligonucleotides having inverted polarity may comprise a single 3'
to 3' linkage at the 3'-most internucleotide linkage. Various
salts, mixed salts and free acid forms of the modified and
non-modified oligonucleotides are also included.
[0040] Preferred modified oligonucleotides may have backbones not
including a phosphorus atom. These backbones may be formed by a
short chain alkyl or cycloalkyl internucleotide linkage, or by one
or more short chain heteroatomic or heterocyclic internucleotide
linkages. These include oligonucleotides having morpholino
linkages; siloxane backbones; sulfide, sulfoxide and sulfone
backbones; formacetyl and thioformacetyl backbones; methylene
formacetyl and thioformacetyl backbones; riboacetyl backbones;
alkene-containing backbones; sulfamate backbones; methyleneimino
and methylenehydrazino backbones; sulfonate and sulfonamide
backbones; amide backbones; and others having mixed N, O, S, and
CH.sub.2 component parts.
[0041] In other oligonucleotide analogs, both the sugar and the
internucleotide linkage of the nucleotides are replaced with novel
groups. The base units are maintained to permit hybridization. One
such oligomeric compound is a peptide nucleic acid, or "PNA." In
PNA compounds, the sugar backbone of an oligonucleotide is replaced
with an amide-containing backbone, such as an aminoethylglycine
backbone.
[0042] Inhibitory compounds of the invention may be formed as
composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide analogs
as described above. Such compounds have also been referred to in
the art as hybrids or gapmers.
[0043] A "coding sequence," or a sequence which "encodes" a
particular protein, is a nucleic acid sequence (or a portion
thereof) which is transcribed (in the case of DNA) or translated
(in the case of mRNA) into a polypeptide in vitro or in vivo when
placed under the control of appropriate regulatory sequences. A
coding sequence can include, but is not limited to, cDNA from
prokaryotic or eukaryotic mRNA, genomic DNA sequences from
prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A
transcription termination sequence will usually be located 3' to
the coding sequence.
[0044] For the purpose of describing the relative position of
nucleotide sequences in a particular nucleic acid molecule
throughout the instant application, such as when a particular
nucleotide sequence is described as being situated "upstream,"
"downstream," "5'" or "3'" relative to another sequence, it is to
be understood that it is the position of the sequences in the
"sense" or "coding" strand of a DNA molecule that is being referred
to, as is conventional in the art.
[0045] "Homology" refers to the percent of identity between at
least two oligonucleotides or polypeptides. The percent identity
between the sequences from one moiety to another can be determined
by techniques known in the art. Homology can be determined by a
direct comparison of the sequence information between two
polypeptide molecules by aligning the sequence information and
using readily available computer programs such as ALIGN, Dayhoff,
M. O. (1978) in Atlas of Protein Sequence and Structure 5:Supp. 3,
National Biomedical Research Foundation, Washington, D.C. Default
parameters can be used for alignment. One alignment program is
BLAST, used with default or manually set parameters. Details of
these programs can be found at the following Internet address:
http://www.ncbi.nlm.gov/cti-bin/BLAST.
[0046] Alternatively, homology can be determined by hybridization
of polynucleotides under conditions which form stable duplexes
between homologous regions, followed by digestion with
single-stranded-specific nuclease(s), and size determination of the
digested fragments. Two DNA, or two polypeptide sequences are
"substantially homologous" to each other when the sequences exhibit
at least about 80%-85%, preferably at least about 90%, and most
preferably at least about 95%-98% sequence identity over a defined
length of the molecules, as determined using the methods above. As
used herein, substantially homologous also refers to sequences
showing complete identity to the specified DNA or polypeptide
sequence. DNA sequences that are substantially homologous can be
identified in a Southern hybridization experiment under, for
example, stringent conditions, as defined for that particular
system. Defining appropriate hybridization conditions is within the
skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning,
supra; Nucleic Acid Hybridization, supra.
[0047] A "functional homologue" or a "functional equivalent" of a
given polypeptide includes molecules derived from the wild-type
polypeptide sequence, as well as recombinantly-produced or
chemically-synthesized polypeptides which function in a manner
similar to the wild-type molecule to achieve a desired result.
Thus, a functional homologue of Bcl-2 encompasses derivatives and
analogues of those polypeptides--including any single or multiple
amino acid additions, substitutions and/or deletions occurring
internally or at the amino or carboxy termini thereof--so long as
integration activity remains.
[0048] "Gene transfer" or "gene delivery" refers to methods or
systems for reliably inserting foreign DNA into host cells. Such
methods can result in transient expression of non-integrated
transferred DNA, extrachromosomal replication and expression of
transferred replicons (e.g., episomes), or integration of
transferred genetic material into the genomic DNA of host
cells.
[0049] By "vector" is meant any genetic element, such as a plasmid,
phage, transposon, cosmid, chromosome, virus, virion, etc., which
can transfer gene sequences into cells and which may or may not
replicate in the host cells. Thus, the term includes cloning and
expression vehicles, as well as viral vectors.
[0050] The term "transfection" is used to refer to the uptake of
foreign DNA by a cell, and a cell has been "transfected" when
exogenous DNA has been introduced inside the cell membrane. A
number of transfection techniques are generally known in the art.
See, e.g., Graham et al., (1973) Virology, 52:456, Sambrook et al.,
(1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor
Laboratories, New York, Davis et al., (1986) Basic Methods in
Molecular Biology, Elsevier, and Chu et al., (1981) Gene 13:197.
Such techniques can be used to introduce one or more exogenous DNA
moieties, such as a nucleotide integration vector and other nucleic
acid molecules, into suitable host cells.
[0051] A "host cell" as used herein may be either a eukaryotic or a
prokaryotic cell. In particular, a host cell could be a yeast cell,
an insect cell, or a mammalian cell which has been transfected with
an exogenous DNA sequence, and the progeny of that cell. It is
understood that the progeny of a single parental cell may not
necessarily be completely identical in morphology or in genomic or
total DNA complement to the original parent, due to natural,
accidental, or deliberate mutation.
[0052] As used herein, the term "cell line" refers to a population
of cells capable of continuous or prolonged growth and division in
vitro. Often, cell lines are clonal populations derived from a
single progenitor cell. It is further known in the art that
spontaneous or induced changes can occur in karyotype during
storage or transfer of such clonal populations. Therefore, cells
derived from the cell line referred to may not be precisely
identical to the ancestral cells or cultures, and the cell line
referred to includes such variants.
[0053] The term "control sequences" refers collectively to promoter
sequences, polyadenylation signals, transcription termination
sequences, upstream regulatory domains, origins of replication,
internal ribosome entry sites ("IRES"), enhancers, and the like,
which collectively provide for the replication, transcription, and
translation of a coding sequence in a recipient cell. Not all of
these control sequences need to be present so long as the selected
coding sequence is capable of being replicated, transcribed, and
translated in an appropriate host cell.
[0054] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured to as to perform
their usual function. Thus, control sequences operably linked to a
coding sequence are capable of effecting the expression of the
coding sequence. The control sequences need not be contiguous with
the coding sequence, so long as they function to direct the
expression thereof. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter sequence
and the coding sequence and the promoter sequence can still be
considered "operably linked" to the coding sequence.
[0055] The term "prodrug" indicates a therapeutic agent that is
prepared in an inactive form that is converted to an active form
(drug) within the body or cells thereof by the action of endogenous
enzymes or other chemicals and/or conditions.
[0056] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
compounds of the invention: i.e., salts that retain the desired
biological activity of the parent compound without adding any
undesired toxicological effects.
2. GENERAL METHODS
[0057] Gene inhibition technologies are often used to modulate
functions such as DNA replication or transcription, RNA
translocation to the site of translation, RNA translation, RNA
splicing, and catalytic activity conducted or aided by the RNA. In
the present invention, the overall effect of such interference with
the function of the target nucleic acid is modulation of the
expression of Bcl-2. This is brought about by the interference of
the single-stranded inhibitory oligonucleotides of the invention
with Bcl-2 mRNA. The interference of the inhibitory
oligonucleotides blocks proper function of the Bcl-2 mRNA, thus
preventing proper expression. This interference is commonly
referred to as "knockdown" of the target nucleic acid. This may
result in the amelioration of disease symptoms or the curing of
various diseases. In the context of this invention, "modulation"
can mean either an increase or a decrease in the expression of a
gene. In the instant invention, inhibition is a preferred form of
modulation of gene expression, and mRNA is a preferred nucleic acid
target.
[0058] Targeting inhibitory compounds to specific nucleic acids is
generally a multistep process. First, a nucleic acid is identified
that participates in a disease state. This nucleic acid is then
sequenced. The nucleic acid may be, for example, a cellular gene or
mRNA whose expression produces a product active in the disease, or
in a nucleic acid molecule from infectious agent such as a virus,
bacterium, or other infectious microbe. In this invention the
target sequence is one encoding Bcl-2. A next step in the targeting
process involves determining potential sites on the target nucleic
acid molecule which are susceptible to interaction with an
inhibitory oligonucleotide. This process also involves evaluating
whether targeting one specific site will modulate the expression of
a nucleic acid more effectively than targeting other specific
sites.
[0059] In the present invention this targeting process may be
conducted according to a variety of methods known in the art.
Additionally, it may be conducted using the method outlined in U.S.
Pat. No. 6,586,180, entitled Directed Antisense Libraries, which is
incorporated herein in its entirety. That application discloses a
procedure that allows the construction of directed antisense
libraries.
[0060] These libraries contain all overlapping fragments spanning
the entire length of the gene of interest. Transcription in vitro
or in vivo of a DNA fragment produces an inhibitory RNA targeted to
the site on the RNA transcript that is encoded by the DNA fragment.
Transcription of the entire DNA fragment library produces all
antisense RNA molecules targeting all positions on the RNA target.
Expression of this library in mammalian cells therefore allows the
identification of effective target sites on the nucleic acid for
use in antisense-mediated gene inhibition.
[0061] The directed libraries generated above may be assayed for
their ability to modulate the expression of target change in vivo
in cultured cells. For such in vivo assays, the antisense library
is transduced into a suitable cell line expressing the gene of
interest. One of skill in the art may appreciate that transfection
conditions may be chosen such that generally only one member of the
library is taken up by each individual host cell. In this way each
fragment of the gene of interest present in the library may be
separately identified, characterized and isolated from other
fragments of the gene of interest.
[0062] In one embodiment of the present invention, each member in
the library is integrated into the chromosome of one cell at the
same locus. This ensures that (1) only one member of the library is
taken up by each individual host cell; (2) each member of the
library will not be lost during cell division; (3) all members of
the library will be expressed at the same level in the host cells.
The result is a group of cells, each expressing a different
inhibitory molecule targeted to a different site on the RNA
transcript of the gene of interest. All target sites are present in
the entire cell population produced. Using suitable detection
methods, cell clones may be identified in which the expression of
the target RNA has been reduced or eliminated. These clones possess
an inhibitory oligonucleotide targeted to a site on the RNA
transcript which allows effective modulation of the gene's
activity. At this point, the plasmid, or a portion thereof encoding
the inhibitory oligonucleotide may be recovered, and its sequence
identified.
[0063] Once specific target sites have been identified,
oligonucleotides are chosen which sufficiently complementary to the
targets to obtain the desired effect. This can be accomplished by
chemically synthesizing suitable oligonucleotides against the
identified targets, delivering them exogenously to cells, and
assaying the effect by standard methods used to measure gene
expression. Alternatively, the oligonucleotides can be incorporated
individually into a suitable vector and re-introduced into cells.
Their effects are assayed using standard methods.
[0064] Thus, the invention includes inhibitory oligonucleotides
directed to three different target regions and to subsequences of
these regions found on nucleic acids encoding Bcl-2 to modulate the
function of the nucleic acid. Each of these target regions contains
many possible inhibitory oligonucleotides, each of which falls
within the scope of this invention. The invention further includes
specific inhibitory oligonucleotides identified from among the
potential inhibitory oligonucleotides for each target sequence,
which are presently preferred for the practice of this
invention.
[0065] A first target region of the Bcl-2 nucleic acids (Region A)
is found in SEQ ID NO: 19 and its sequence variant SEQ ID NO: 36. A
subsequence of this first target region (SEQ ID NO: 15, and its
corresponding sequence variant) provided a particularly high number
of inhibitory oligonucleotides in the screening procedure. Region A
is about 40 nucleobases in length, and the Region A subsequence
represented by SEQ ID NO: 15 is about 30 nucleobases in length.
Oligonucleotides complementary to a portion of the target region of
Region A or to SEQ ID NO: 15 are inhibitory oligonucleotides which
may modulate the expression of Bcl-2. These oligonucleotides may
preferably be between from about 8 to about 30 bases in length,
more preferably about 10 to about 26 nucleobases in length, and
most preferably about 14 to about 20 nucleobases in length. Such
oligonucleotides which are complementary to at least a portion of
Region A or to at least a portion of SEQ ID NO. 15 include but are
not limited to, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14, SEQ
ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, which
are useful as inhibitory oligonucleotides in the practice of the
invention. The invention further includes oligonucleotides
comprising at least 8 contiguous nucleobases of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO: 7, SEQ ID NO: 14, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO: 24 and
SEQ ID NO: 25, for use as inhibitory oligonucleotides.
[0066] A second target region of the Bcl-2 nucleic acids (Region B)
is found in SEQ ID NO: 20 and its sequence variant SEQ ID NO: 37. A
preferred subsequence of Region B for derivation of inhibitory
oligonucleotides is SEQ ID NO: 16 and its corresponding sequence
variant. Region B is about 62 nucleobases in length, and SEQ ID NO:
16 is about 14 nucleobases in length. Oligonucleotides
complementary to at least a portion of the target region of Region
B or to at least a portion of SEQ ID NO: 16 are inhibitory
oligonucleotides which may modulate the expression of Bcl-2 and are
typically about 10-26 nucleobases in length. These oligonucleotides
may preferably be about 8 to about 14 nucleobases in length, more
preferably about 10 to about 14 nucleobases in length, and most
preferably about 12 to about 14 nucleobases in length. Such
oligonucleotides which are complementary to Region B or to SEQ. ID.
NO: 16 include, but are not limited to, SEQ ID NO: 8, SEQ ID NO: 9,
SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ
ID NO: 30, which are useful as inhibitory oligonucleotides in the
practice of the invention. The invention further includes
oligonucleotides comprising at least 8 contiguous nucleobases of
SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID
NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30 for use as inhibitory
oligonucleotides.
[0067] A third target region of the Bcl-2 nucleic acids (Region C)
is found in SEQ ID NO: 21. A preferred subsequence of SEQ ID NO: 21
for derivation of inhibitory oligonucleotides is SEQ ID NO: 17.
Region C is about 23 nucleobases in length. SEQ ID NO: 17 is about
17 nucleobases in length. Oligonucleotides complementary to at
least a portion of the target region of Region C or to at least a
portion of SEQ ID NO: 17 are inhibitory oligonucleotides which may
modulate the expression of Bcl-2 and are typically about 100-23
nucleobases in length. These oligonucleotides may preferably be
between about 8 to about 17 nucleobases in length, more preferably
about 10 to about 17 nucleobases in length, and most preferably
about 12 to about 14 nucleobases in length. Such oligonucleotides
which are complementary to Region C or to SEQ ID NO: 17 include,
but are not limited to, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:
12, SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and
SEQ ID NO: 34, which are useful as inhibitory oligonucleotides in
the practice of the invention. The invention further includes
oligonucleotides comprising least 8 contiguous nucleobases of SEQ
ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:
31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 for use as
inhibitory oligonucleotides.
[0068] The oligonucleotides of the invention may be incorporated
into pharmaceutical compositions. The oligonucleotides may
additionally be used in methods to modulate the expression of Bcl-2
in a cell or tissue. Such methods may use the oligonucleotides
alone, or they may use the pharmaceutical compositions of the
invention.
[0069] Inhibitory oligonucleotide compounds are commonly used as
research reagents and in diagnostics. Due to their ability to
inhibit gene expression with specificity, such oligonucleotides are
often used by those of ordinary skill to elucidate the function of
particular genes. Inhibitory oligonucleotide compounds are also
used to distinguish the functions of various members of a
biological pathway.
[0070] The specificity and sensitivity of inhibitory
oligonucleotide technology is also harnessed by those skilled in
the art for therapeutic uses. One type of this technology is
antisense oligonucleotides. Antisense oligonucleotides have been
employed as therapeutic moieties in the treatment of disease states
in animals and man. Other drugs, including ribozymes, have been
safely and effectively administered to humans, and numerous
clinical trials are presently underway. As useful therapeutic
modalities, inhibitory oligonucleotides can be configured to be
useful in treatment regimes for treatment of cells, tissues, and
animals, especially humans.
[0071] The inhibitory compounds of this invention may be
conveniently and routinely made through techniques such as solid
phase synthesis. Equipment for such synthesis is sold by several
vendors including, for example, Applied Biosystems, (Foster City,
Calif.). Any other means for such synthesis known in the art may
additionally or alternatively be employed. It is well known to use
similar techniques to prepare oligonucleotides such as the
phosphorothioates and alkylated derivatives.
[0072] The invention encompasses any pharmaceutically acceptable
salts, esters, salts of such esters, or any other compounds which,
upon administration to an organism such as a human, are capable of
providing (directly or indirectly) the biologically active
inhibitory oligonucleotides of the invention. Accordingly, for
example, the disclosure is also drawn to prodrugs, and other
bioequivalents. The compounds of the invention may also be admixed,
encapsulated, conjugated or otherwise associated with other
molecules, molecule structures or mixtures of compounds, as for
example, liposomes, receptor targeted molecules, oral, topical and
other formulations, for assisting in uptake, distribution and/or
absorption.
[0073] The inhibitory compounds of the present invention can be
used as diagnostics, therapeutics, prophylaxis, and as research
reagents and kits. For therapeutics, an organism, such as a human,
having a disease or disorder which can be treated by modulating the
expression of Bcl-2 is treated by administering inhibitory
compounds in accordance with this invention. The compounds of the
invention can be utilized in pharmaceutical compositions by adding
an effective amount of the inhibitory compound to a suitable
pharmaceutically acceptable diluent or carrier. The inhibitory
compounds and methods of the invention may also be useful to
prevent or delay infection, inflammation, or tumor formation, for
example.
[0074] The inhibitory compounds of the invention are useful for
research and diagnostics because these compounds hybridize to
nucleic acids encoding Bcl-2. Hybridization of the inhibitory
oligonucleotides of the invention with a nucleic acid encoding
Bcl-2 can be detected by means known in the art. Such means may
include conjugating an enzyme to the oligonucleotides,
radiolabeling the oligonucleotide, or using any other suitable
detection means. Kits using such detection means for detecting the
level of Bcl-2 present in a sample may also be prepared.
[0075] The pharmaceutical compositions of the present invention may
be administered in a number of ways depending upon whether local or
systemic treatment is desired and upon the area to be treated.
Administration may be topical (including ophthalmic and to mucous
membranes), pulmonary, e.g., by inhalation or insufflation of
powders or aerosols, including by nebulizers; and tracheal,
intranasal, epidermal, transdermal, oral, or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal, or intramuscular injection or infusion, as well as
intracranial (e.g., intrathecal or intraventricular)
administration. Oligonucleotides with at least one
2'-O-methoxyethyl modification are believed to be particularly
useful for oral administration.
[0076] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids, and powders.
Conventional pharmaceutical carriers, aqueous bases, powder bases
or oil bases, thickeners and the like may be necessary or
desirable. Topical formulations include those in which the
oligonucleotides of the invention are in admixture with a topical
delivery agent such as lipids, liposomes, fatty acids, fatty acid
esters, steroids, chelating agents and surfactants. Preferred
lipids and liposomes may be neutral, negative, and cationic.
Oligonucleotides of the invention may be encapsulated within
liposomes or may form complexes thereto, such as cationic
liposomes. Alternatively, oligonucleotides may be complexed to
lipids, including cationic lipids.
3. EXAMPLES
Example 1
[0077] In a first example, an anti-Bcl-2 antisense oligonucleotide
library was created using the methods of the invention outlined
above. This antisense plasmid library contained nucleotides likely
targeting every nucleotide position in the Bcl-2 RNA transcript.
Nucleotide sequences which repressed the expression of Bcl-2 were
isolated from this library by introducing the plasmid library into
mammalian cells. The mammalian cells expressed the anti-Bcl-2
sequences upon the addition of an inducer, thus producing
anti-Bcl-2 RNAs. Since inhibition of Bcl-2 is generally detrimental
to cells, expression of the anti-Bcl-2 sequences was induced when
the cells were ready to be sorted. In this example, tetracycline
was used as the inducer. Other suitable systems of inducers and
inducible promoters are known to those of skill in the art.
[0078] Following induction and expression of the anti-Bcl-2
sequences, Bcl-2 negative cells were sorted out from the others
using a fluorescein-activated cell sorter. The anti-Bcl-2
nucleotide sequences from the Bcl-2 negative cells are then
retrieved and sequenced. These anti-Bcl-2 sequences may be useful
as therapeutic agents for diseases including cancer.
Example 2
[0079] The cell line A-2780R is derived from a human ovarian
carcinoma and is known to express the Bcl-2 protein. When
subcutaneously implanted into an athymic nude (immunodeficient)
mouse host, A-2780R readily forms a progressively growing tumor
mass. This provides a convenient in vivo model in which
Bcl-2-active anticancer agents can be evaluated for therapeutic
efficacy. In order to demonstrate the in vivo anticancer efficacy
of a representative inhibitory oligonucleotide of the invention,
athymic nude mice were subcutaneously implanted with A-2780R
tumors. The tumor-bearing mice were then treated with G3139
(positive control: a Bcl-2 antisense oligonucleotide being
developed by Genta, Inc.) or with SEQ ID NO: 32 (in DNA format).
Tumor growth in these two treatment groups was compared to an
untreated control group to determine therapeutic efficacy.
Experimental details were as follows:
[0080] Female nude Balb/c mice (nu/nu), approximately 5 weeks old
(Animal Technologies, Fremont, Calif.) were each implanted with
10.sup.7 A-2780R tumor cells by subcutaneous (SC) injection of 0.1
ml of inoculum into the right hind flank. The tumor cell inoculum
was prepared in sterile DME/F-12 medium+10% fetal bovine serum, at
a density of 1.0.times.10.sup.8 cells/ml. When tumors were
approximately 6 mm.times.6 mm in size (about 110 mg), the animals
were group-matched into treatment and control groups, such that the
mean tumor mass was normalized on a group-by-group basis (study day
1). Each group consisted of 4 animals. Mean body weight was about
20 gm at this time.
[0081] Solutions for injection of the phosphorothioate DNA
oligonucleotides SEQ ID NO: 32 (Applied Biosystems, Salt Lake City,
Utah) and G3139 (GENASENSE.TM., TriLink BioTechnologies, Inc., San
Diego, Calif.) were prepared in phosphate buffered saline, pH 7.2
(PBS) at a final DNA concentration of 1.0 mg/ml. On study days 1,
2, and 3 each animal was administered a 0.2 ml volume of one of the
solutions by IV injection into the lateral tail vein (200 .mu.g of
DNA or 10 mg DNA/Kg per dose). Untreated control animals received a
sham injection of PBS only in the same manner as the
oligonucleotide treatment groups.
[0082] On study days 1, 4, 7 and 9 the animals were weighed. Tumor
dimensions (length and width) were measured every other day and
converted to tumor mass (mg) using the formula: Tumor
Mass=L.sup.2.times.W/2. The resulting tumor mass values were then
averaged for each study group for each study day and were plotted
against time. The results of the study as they relate to the effect
of the oligonucleotides on tumor mass are shown in FIG. 2.
[0083] The tumor growth inhibitory activity of the two DNA
oligonucleotides was summarized by calculating the percentage of
tumor growth inhibition as compared to the untreated control group
for the study day 4, 7 and 9 timepoints. Percent tumor growth
inhibition was calculated using the formula: % Tumor Growth
Inhibition=100-[(mean treated tumor wt./mean control tumor
wt.).times.100]. Tumor growth inhibitory activity is summarized in
the following Table: TABLE-US-00001 Experimental DNA Dose % Tumor
Growth Inhibition Treatment (mg/Kg) Day 4 Day 7 Day 9 Untreated 0 0
0 0 Control G3139 10 28.9 32.7 24.0 SEQ ID 10 52.5 67.9 72.0 NO:
32
[0084] Although both antisense Bcl-2 oligonucleotides were found to
be inhibitory to the growth of A-2780R tumors, SEQ ID NO: 32 was
approximately 2-fold more effective than the positive control
(G3139) at each study timepoint. Treatment with SEQ ID NO: 32
resulted in a maximal tumor growth inhibition value of 72%, which
occurred on study day 9. For animals treated with the positive
control G3139, maximal tumor growth inhibition occurred on study
day 7 and was found to be only 32.7%.
[0085] The effect of treatment on body weight was also evaluated.
Weight loss in response to treatment is a general indicator of
systemic toxicity. Percent change in body weight was calculated for
each group at each study timepoint using the formula: % Change in
Body Weight=[(mean body wt., day X-mean body wt., day 1)/mean body
wt., day 1].times.100. Both oligonucleotides were found to be
well-tolerated by the animals through the duration of the study, as
no weight loss occurred in any of the study groups.
Example 3
[0086] The cell line PC-3 is derived from a human prostate
adenocarcinoma and is known to express the Bcl-2 protein. This cell
line is therefore well suited for use in in vitro transfection
studies for the evaluation of antisense Bcl-2 oligonucleotides. In
order to demonstrate the in vitro cytotoxic or growth inhibitory
activity of a representative inhibitory oligonucleotide of the
invention against Bcl-2-expressing malignancies, PC-3 cells were
transfected in vitro using SEQ ID NO: 5 and SEQ ID NO: 32 in DNA
format. The antisense Bcl-2 DNA oligonucleotide G3139 was used as a
positive control. Following transfection the effect on cell density
was measured and cytotoxic or growth-inhibitory activity was
determined by comparison to untreated control cells. Experimental
details were as follows:
[0087] The DNA oligonucleotides evaluated in the study were SEQ ID
NO: 5 (Applied Biosystems, Salt Lake City, Utah), SEQ ID NO: 32
(Applied Biosystems, Salt Lake City, Utah), G3139 (MWG-Biotech,
High Point, N.C.) and a DNA oligonucleotide derived from the Bcl-2
gene but outside of target Regions A, B and C.
[0088] The four antisense Bcl-2 DNA oligonucleotides were each
initially diluted to a concentration of 4.0 .mu.M using serum-free
tissue culture medium (MEM/EBSS). Each oligonucleotide solution was
then complexed to the transfection agent Lipofectamine 2000
(Invitrogen, life technologies) by addition of the DNA solutions to
a separate set of Lipofectamine 2000 solutions (also prepared in
serum-free tissue culture medium), such that the coupling ratio of
DNA (in .mu.g):Lipofectamine 2000 (in .mu.l) was 1:2.5. A separate
Lipofectamine 2000 control solution was also prepared, which
contained Lipofectamine 2000 at an equivalent concentration,
diluted in serum-free medium without DNA (the Lipofectamine
control). In the same manner a second set of DNA solutions was
prepared in serum-free medium, but which did not contain the
Lipofectamine 2000 transfection agent (DNA control). The resulting
solutions were mixed and incubated at room temperature for 20 min.
to allow the complexation interaction to reach equilibrium. Each of
the resulting nine solutions was serially diluted in two-fold steps
using serum-free tissue culture medium. A suspension of PC-3 cells
(American Type Culture Collection, Manassas, Va.) was prepared at a
density of 1.times.10.sup.6 cells/ml in tissue culture medium
(MEM/EBSS) supplemented with 20% fetal bovine serum. Each of the
DNA-Lipofectamine 2000 complex solutions, uncomplexed DNA control
solutions and Lipofectamine 2000 control solutions (50 .mu.l) were
placed into the wells of a 96-well assay plate, followed by 50
.mu.l of PC-3 cell suspension (50,000 cells). Cell control wells
contained 50,000 PC-3 cells in 100 .mu.l of tissue culture medium
supplemented with 10% fetal bovine serum. All wells were prepared
in duplicate. The assay plates were then incubated at 37.degree. C.
in an atmosphere containing 5% CO.sub.2 (95% air) and humidity for
72 hours.
[0089] Following the 72 hour incubation, 20 .mu.l of MTS/PES
solution (Promega CellTiter 96 AQ One Solution Cell Proliferation
Assay, No. G-3580) was added to each well of the plates. The assay
plates were incubated for an additional 2 hours at 37.degree. C. as
described above. Using a 96-well microplate reader the absorbance
of each well was measured at 490 nm. The instrument was zeroed
using wells containing 100 .mu.l of tissue culture medium
supplemented with 10% fetal bovine serum and 20 .mu.l of MTS/PES
solution. The mean absorbance value was calculated for each
duplicate pair of wells, and the resulting values were then used to
calculate % growth inhibition using the formula: % Growth
Inhibition=(1-[Abs. Transfected Test Group/Abs. Cell
Control]).times.100. Dose response curves were plotted using a
linear y-axis scale for % cell growth inhibition and a logarithmic
x-axis scale for DNA concentration. The cell growth inhibitory
activity was compared between each transfection group by
determining the 50% inhibitory concentration (IC.sub.50) value for
each group. The IC.sub.50 value is defined as the DNA concentration
which resulted in the inhibition of growth or death of 50% of the
cells as compared to the untreated control (cell control). The
results are shown in FIG. 3
[0090] The corresponding IC.sub.50 values for each transfection
group are shown in the following Table: TABLE-US-00002 Experimental
Treatment IC.sub.50 (DNA Conc.) Lipofectamine Alone (control)
>1000 nM equiv. G3139 DNA + Lipofectamine 280 nM SEQ ID NO: 5 +
Lipofectamine 233 nM SEQ ID NO: 32 + Lipofectamine 184 nM
Non-Region A, B, C oligo + 503 nM Lipofectamine
[0091] When complexed to the Lipofectamine 2000 transfection agent,
all four antisense Bcl-2 DNA oligonucleotides resulted in
inhibition of PC-3 cell growth at 72 hours following initiation of
transfection. SEQ ID NO: 32 (Region C) was found to be the most
active, having an IC.sub.50 value of 184 nM, which was 1.52-fold
more active than the G3139 positive control (Region A,
IC.sub.50=280 nM). The IC.sub.50 value for SEQ ID NO: 5 (Region A)
also indicated a high level of inhibitory activity. The non-Region
A, B, C oligonucleotide, although active, was substantially less
inhibitory than the oligonucleotides derived from the target
regions of the invention. The Lipofectamine 2000 control was only
minimally toxic to the cells at the highest concentration. Cells
treated with DNA alone (in the absence of Lipofectamine 2000
transfection agent) showed little or no inhibition of growth.
[0092] The present invention may be embodied in other specific
forms without departing from its structures, methods, or other
essential characteristics as broadly described herein and claimed
hereinafter. The described embodiments are to be considered in all
respects only as illustrative, and not restrictive. The scope of
the invention is, therefore, indicated by the appended claims,
rather than by the foregoing description. All changes that come
within the meaning and range of equivalency of the claims are to be
embraced within their scope.
Sequence CWU 1
1
38 1 14 DNA Homo sapiens 1 agcgtgcgcc atcc 14 2 14 DNA Homo sapiens
2 cgccatcctt ccca 14 3 14 DNA Homo sapiens 3 atccttccca gagg 14 4
14 DNA Homo sapiens 4 cccagaggaa aagc 14 5 18 DNA Homo sapiens 5
ccttcccaga ggaaaagc 18 6 14 DNA Homo sapiens 6 ccttcccaga ggaa 14 7
14 DNA Homo sapiens 7 catccttccc agag 14 8 14 DNA Homo sapiens 8
gggagaagtc gtcg 14 9 14 DNA Homo sapiens 9 cggcttggcg gagg 14 10 14
DNA Homo sapiens 10 ccccgcgcgg tgaa 14 11 14 DNA Homo sapiens 11
ccgcgcggtg aagg 14 12 14 DNA Homo sapiens 12 cgcgcggtga aggg 14 13
13 DNA Homo sapiens 13 gcgcggtgaa ggg 13 14 14 DNA Homo sapiens 14
tcccagagga aaag 14 15 30 DNA Homo sapiens 15 gcttttcctc tgggaaggat
ggcgcacgct 30 16 14 DNA Homo sapiens 16 cgacgacttc tccc 14 17 17
DNA Homo sapiens 17 cccttcaccg cgcgggg 17 18 931 DNA Homo sapiens
18 gctggggcga gaggtgccgt tggcccccgt tgcttttcct ctgggaagga
tggcgcacgc 60 tgggagaacg gggtacgaca accgggagat agtgatgaag
tacatccatt ataagctgtc 120 gcagaggggc tacgagtggg atgcgggaga
tgtgggcgcc gcgcccccgg gggccgcccc 180 cgcaccgggc atcttctcct
cccagcccgg gcacacgccc catccagccg catcccgcga 240 cccggtcgcc
aggacctcgc cgctgcagac cccggctgcc cccggcgccg ccgcggggcc 300
tgcgctcagc ccggtgccac ctgtggtcca cctggccctc cgccaagccg gcgacgactt
360 ctcccgccgc taccgcggcg acttcgccga gatgtccagc cagctgcacc
tgacgccctt 420 caccgcgcgg ggacgctttg ccacggtggt ggaggagctc
ttcagggacg gggtgaactg 480 ggggaggatt gtggccttct ttgagttcgg
tggggtcatg tgtgtggaga gcgtcaaccg 540 ggagatgtcg cccctggtgg
acaacatcgc cctgtggatg actgagtacc tgaaccggca 600 cctgcacacc
tggatccagg ataacggagg ctgggatgcc tttgtggaac tgtacggccc 660
cagcatgcgg cctctgtttg atttctcctg gctgtctctg aagactctgc tcagtttggc
720 cctggtggga gcttgcatca ccctgggtgc ctatctgagc cacaagtgaa
gtcaacatgc 780 ctgccccaaa caaatatgca aaaggttcac taaagcagta
gaaataatat gcattgtcag 840 tgatgtacca tgaaacaaag ctgcaggctg
tttaagaaaa aataacacac atataaacat 900 cacacacaca gacagacaca
cacacacaca a 931 19 40 DNA Homo sapiens 19 gcttttcctc tgggaaggat
ggcgcacgct gggagaacgg 40 20 62 DNA Homo sapiens 20 cctccgccaa
gccggcgacg acttctcccg ccgctaccgc ggcgacttcg ccgagatgtc 60 ca 62 21
23 DNA Homo sapiens 21 gacgcccttc accgcgcggg gac 23 22 14 DNA Homo
sapiens 22 gccatccttc ccag 14 23 14 DNA Homo sapiens 23 cgtgcgccat
cctt 14 24 14 DNA Homo sapiens 24 gcgtgcgcca tcct 14 25 14 DNA Homo
sapiens 25 ccgttctccc agcg 14 26 14 DNA Homo sapiens 26 gcggtagcgg
cggg 14 27 14 DNA Homo sapiens 27 cgccgcggta gcgg 14 28 14 DNA Homo
sapiens 28 ggacatctcg gcga 14 29 20 DNA Homo sapiens 29 agaagtcgtc
gccggcttgg 20 30 20 DNA Homo sapiens 30 tggacatctc ggcgaagtcg 20 31
20 DNA Homo sapiens 31 cccgcgcggt gaagggcgtc 20 32 18 DNA Homo
sapiens 32 ccccgcgcgg tgaagggc 18 33 14 DNA Homo sapiens 33
cccgcgcggt gaag 14 34 14 DNA Homo sapiens 34 gtccccgcgc ggtg 14 35
931 DNA Homo sapiens 35 gctggggcga gaggtgccgt tggcccccgt tacttttcct
ctgggaaata tggcgcacgc 60 tgggagaaca gggtacgaca accgggagat
agtgatgaag tacatccatt ataagctgtc 120 gcagaggggc tacgagtggg
atgcgggaga tgtgggcgcc gcgcccccgg gggccgcccc 180 cgcgccgggc
atcttctcct cgcagcccgg gcacacgccc catacagccg catcccggga 240
cccggtcgcc aggacctcgc cgctgcagac cccggctgcc cccggcgccg ccgcggggcc
300 tgcgctcagc ccggtgccac ctgtggtcca cctgaccctc cgccaggccg
gcgacgactt 360 ctcccgccgc taccgccgcg acttcgccga gatgtccagg
cagctgcacc tgacgccctt 420 caccgcgcgg ggacgctttg ccacggtggt
ggaggagctc ttcagggacg gggtgaactg 480 ggggaggatt gtggccttct
ttgagttcgg tggggtcatg tgtgtggaga gcgtcaaccg 540 ggagatgtcg
cccctggtgg acaacatcgc cctgtggatg actgagtacc tgaaccggca 600
cctgcacacc tggatccagg ataacggagg ctgggatgcc tttgtggaac tgtacggccc
660 cagcatgcgg cctctgtttg atttctcctg gctgtctctg aagactctgc
tcagtttggc 720 cctggtggga gcttgcatca ccctgggtgc ctatctgggc
cacaagtgaa gtcaacatgc 780 ctgccccaaa caaatatgca aaaggttcac
taaagcagta gaaataatat gcattgtcag 840 tgatgttcca tgaaacaaag
ctgcaggctg tttaagaaaa aataacacac atataaacat 900 cacacacaca
gacagacaca cacacacaca a 931 36 40 DNA Homo sapiens 36 acttttcctc
tgggaaatat ggcgcacgct gggagaacag 40 37 62 DNA Homo sapiens 37
cctccgccag gccggcgacg acttctcccg ccgctaccgc cgcgacttcg ccgagatgtc
60 ca 62 38 480 DNA Homo sapiens 38 gttggccccc gttgcttttc
ctctgggaag gatggcgcac gctgggagaa cggggtacga 60 caaccgggag
atagtgatga agtacatcca ttataagctg tcgcagaggg gctacgagtg 120
ggatgcggga gatgtgggcg ccgcgccccc gggggccgcc cccgcaccgg gcatcttctc
180 ctcccagccc gggcacacgc cccatccagc cgcatcccgc gacccggtcg
ccaggacctc 240 gccgctgcag accccggctg cccccggcgc cgccgcgggg
cctgcgctca gcccggtgcc 300 acctgtggtc cacctggccc tccgccaagc
cggcgacgac ttctcccgcc gctaccgcgg 360 cgacttcgcc gagatgtcca
gccagctgca cctgacgccc ttcaccgcgc ggggacgctt 420 tgccacggtg
gtggaggagc tcttcaggga cggggtgaac tgggggagga ttgtggcctt 480
* * * * *
References